De novo dna cytosine methyltransferase genes, polypeptides and uses thereof

ABSTRACT

De novo DNA cytosine methyltransferase polynucleotides and polypeptides and methods for producing said polypeptides are disclosed. Also disclosed are methods for utilizing de novo DNA cytosine methyltransferase polynucleotides and polypeptides in diagnostic assays, for an in vitro DNA methylation application and therapeutic applications such as the treatment of neoplastic disorders.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularbiology, developmental biology, cancer biology and medical therapeutics.Specifically, the present invention relates to novel DNA cytosinemethyltransferases. More specifically, isolated nucleic acid moleculesare provided encoding mouse Dnmt3a and Dnmt3b and human DNMT3A andDNMT3B de novo DNA cytosine methyltransferase genes. Dnmt3a and Dnmt3bmouse and DNMT3A and DNMT3B human polypeptides are also provided, as arevectors, host cells and recombinant methods for producing the same. Theinvention further relates to an in vitro method for cytosine C5methylation. Also provided is a diagnostic method for neoplasticdisorders, and methods of gene therapy using the polynucleotides of theinvention.

2. Related Art

Methylation at the C-5 position of cytosine predominantly in CpGdinucleotides is the major form of DNA modification in vertebrate andinvertebrate animals, plants, and fungi. Two distinctive enzymaticactivities have been shown to be present in these organisms. The de novoDNA cytosine methyltransferase, whose expression is tightly regulated indevelopment, methylates unmodified CpG sites to establish tissue orgene-specific methylation patterns. The maintenance methyltransferasetransfers a methyl group to cytosine in hemi-methylated CpG sites inreplicated DNA, thus functioning, to maintain clonal inheritance of theexisting methylation patterns.

De novo methylation of genomic DNA is a developmentally regulatedprocess (Jähaner, D. and Jaenish, R., “DNA Methylation in EarlyMammalian Development,” In DNA Methylation: Biochemistry and BiologicalSignificance, Razin, A. et al., eds., Springer-Verlag (1984) pp. 189-219and Razin, A., and Cedar, H., “DNA Methylation and Embryogenesis,” inDNA Methylation: Molecular Biology and Biological Significance, Jost.,J. P. et al., eds., Birkhäuser Verlag, Basel, Switzerland (1993) pp.343-357). It plays a pivotal role in the establishment ofparental-specific methylation patterns of imprinted genes (Chaillet. J.R. et al., Cell 66:77-83 (1991); Stöger, R. et al, Cell 73:61-71 (1993);Brandeis, M. et al. EMBO J. 12:3669-3677 (1993); Tremblay, K. D. et al.,Nature Genet. 9:407-413 (1995); and Tucker, K. L. et al., Genes Dev.10:1008-1020 (1996)), and in the regulation of X chromosome inactivationin mammals (Brockdoff, N. “Convergent Themes in X ChromosomeInactivation and Autosomal Imprinting,” in Genomic Imprinting: Frontiersin Molecular Biology, Reik, W. and Sorani, A. eds., IRL Press Oxford(1997) pp. 191-210; Ariel, M. et al., Nature Genet. 9:312-315 (1995);and Zucotti, M. and Monk, M. Nature Genet. 9:316-320 (1995)).

Thus, C5 methylation is a tightly regulated biological process importantin the control of gene regulation. Additionally, aberrant de novomethylation can lead to undesirable consequences. For example, de novomethylation of growth regulatory genes in somatic tissues is associatedwith tumorigenesis in humans (Laird, P. W. and Jaenisch, R. Ann. Rev.Genet. 30:441-464 (1996); Baylin, S. B. et al., Adv. Cancer. Res.72:141-196 (1998); and Jones, P. A. and Gonzalgo, M. L. Proc. Natl.Acad. Sci. USA 94:2103-2105 (1997)).

The gene encoding the major maintenance methyltransferase, Dnmt1, wasfirst cloned in mice (Bestor. T. H. et al., J. Mol. Biol. 203:971-983(1988), and the homologous genes were subsequently cloned from a numberof organisms, including Arabidoposis, sea urchin, chick, and human.Dnmt1 is expressed ubiquitously in human and mouse tissues. Targeteddisruption of Dnmt1 results in a genome-wide loss of cytosinemethylation and embryonic lethality (Li et al., 1992). Interestingly,Dnmt1 is dispensable for the survival and growth of the embryonic stemcells, but appears to be required for the proliferation ofdifferentiated somatic cells (Lei et al., 1996). Although it has beenshown that the enzyme encoded by Dnmt1 can methylate DNA de novo invitro (Bestor, 1992), there is no evidence that Dnmt1 is directlyinvolved in de novo methylation in normal development. Dnmt1 appears tofunction primarily as a maintenance methyltransferase because of itsstrong preference for hemi-methylated DNA and direct association withnewly replicated DNA (Leonhardt, H. et al., Cell 71:865-873 (1992)).Additionally, ES cells homozygous for a null mutation of Dnmt1 canmethylate newly integrated retroviral DNA, suggesting that Dnmt1 is notrequired for de novo methylation and an independently encoded de novoDNA cytosine methyltransferase is present in mammalian cells (Lei etal., 1996).

Various methods of disrupting Dnmt1 protein activity are known to thoseskilled in the art. For example, see PCT Publication No. WO92/06985,wherein mechanism based inhibitors are discussed. Applications involvingantisense technology are also known; U.S. Pat. No. 5,578,716 disclosesthe use of antisense oligonucleotides to inhibit Dnmt1 activity, andSzyf et al., J. Biol. Chem. 267: 12831-12836, 1992, demonstrates thatmyogenic differentiation can be affected through the antisenseinhibition of Dnmt1 protein activity.

Thus, while there is a significant amount of knowledge in the artregarding the maintenance C5 methyltransferase (Dnmt1), there is noinformation regarding nucleic acid or protein structure and expressionor enzymatic properties of the de novo C5 methyltransferase in mammals.

SUMMARY OF THE INVENTION

A first aspect of the invention provides novel de novo DNA cytosinemethyltransferase nucleic acids and polypeptides that are not availablein the art. A second aspect of the invention relates to de novo DNAcytosine methyltransferase recombinant materials and methods for theirproduction. A third aspect of the invention relates to the production ofrecombinant de novo DNA cytosine methyltransferase polypeptides. Afourth aspect of the invention relates to methods for using such de novoDNA cytosine methyltransferase polypeptides and polynucleotides. Suchuses include the treatment of neoplastic disorders, among others. Yetanother aspect of the invention relates to diagnostic assays for thedetection of diseases associated with inappropriate de novo DNA cytosinemethyltransferase activity or levels and mutations in de novo DNAcytosine methyltransferases that might lead to neoplastic disorders.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1D shows the nucleotide sequences of mouse Dnmt3a and Dnmt3b andhuman DNMT3A and DNMT3B genes, respectively.

FIG. 2A-2D shows the deduced amino acid sequence of mouse Dnmt3a andDnmt3b and human DNMT3A and DNMT3B genes, respectively. Sequences arepresented in single letter amino acid code.

FIG. 3A shows a comparison of mouse Dnmt3a and Dnmt3b amino acidsequences, and FIG. 3B presents a comparison of the protein sequences ofhuman DNMT3A and DNMT3B1.

FIG. 4A presents a schematic comparison of mouse Dnmt1, Dnmt2, Dnmt3aand Dnmt3b protein structures. FIG. 4B presents a schematic of theDNMT3A, DNMT3B and zebrafish Zmt3 proteins. FIGS. 4C and 4D present aschematic of the human DNMT3B gene organization and exon/intron junctionsequences.

FIG. 5A presents a comparison of highly conserved protein structuralmotifs for eukaryotic and prokaryotic C5 methyltransferase. FIG. 5Bpresents a sequence alignment of the C-rich domain of vertebrate DNMT3proteins and the X-lined ATRX gene. FIG. 5C presents a non-rootedphylogenic tree of methyltransferase proteins.

FIG. 6A-6C demonstrates the expression of Dnmt3a and Dnmt3b in mouseadult tissues, embryos, and ES cells by northern blot.

FIG. 7A-7D demonstrates in vitro methyltransferase activities of mouseDnmt3a and Dnmt3b proteins.

FIG. 8 demonstrates in vitro analysis of de novo and maintenanceactivities of Dnmt3a, Dnmt3b1 and Dnmt3b2 proteins.

FIG. 9 presents Northern blot expression analysis of DNMT3A and DNMT3B.

FIG. 10 presents DNMT3 Northern Blot expression analysis of DNMT3A andDNMT3B in human tumor cell lines.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

In the description that follows, a number of terms used in recombinantDNA technology are utilized extensively. In order to provide a clear andconsistent understanding of the specification and claims, including thescope to be given such terms, the following definitions are provided.

Cloning vector: A plasmid or phage DNA or other DNA sequence which isable to replicate autonomously in a host cell, and which ischaracterized by one or a small number of restriction endonucleaserecognition sites at which such DNA sequences may be cut in adeterminable fashion without loss of an essential biological function ofthe vector, and into which a DNA fragment may be spliced in order tobring about its replication and cloning. The cloning vector may furthercontain a marker suitable for use in the identification of cellstransformed with the cloning vector. Markers, for example, providetetracycline resistance or ampicillin resistance.

Expression vector: A vector similar to a cloning vector but which iscapable of enhancing the expression of a gene which has been cloned intoit, after transformation into a host. The cloned gene is usually placedunder the control of (i.e., operably linked to) certain controlsequences such as promoter sequences. Promoter sequences may be eitherconstitutive or inducible.

Recombinant Host: According to the invention, a recombinant host may beany prokaryotic or eukaryotic host cell which contains the desiredcloned genes on an expression vector or cloning vector. This term isalso meant to include those prokaryotic or eukaryotic cells that havebeen genetically engineered to contain the desired gene(s) in thechromosome or genome of that organism. For examples of such hosts, seeSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).Preferred recombinant hosts are eukaryotic cells transformed with theDNA construct of the invention. More specifically, mammalian cells arepreferred.

Recombinant vector: Any cloning vector or expression vector whichcontains the desired cloned gene(s).

Host Animal: Transgenic animals, all of whose germ and somatic cellscontain the DNA construct of the invention. Such transgenic animals arein general vertebrates. Preferred Host Animals are mammals such asnon-human primates, mice, sheep, pigs, cattle, goats, guinea pigs,rodents, e.g. rats, and the like. The term Host Animal also includesanimals in all stages of development, including embryonic and fetalstages.

Promoter: A DNA sequence generally described as the 5′ region of a gene,located proximal to the start codon. The transcription of an adjacentgene(s) is initiated at the promoter region. If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. According to the invention, preferred promoters areheterologous to the de novo DNA cytosine methyltransferase genes, thatis, the promoters do not drive expression of the gene in a mouse orhuman. Such promoters include the CMV promoter (InVitrogen, San Diego,Calif.), the SV40, MMTV, and hMTIIa promoters (U.S. Pat. No. 5,457,034),the HSV-1 4/5 promoter (U.S. Pat. No. 5,501,979), and the earlyintermediate HCMV promoter (WO92/17581). In one emdodiment, it ispreferred that the promoter is tissue-specific, that is, it is inducedselectively in a specific tissue. Also, tissue-specific enhancerelements may be employed. Additionally, such promoters may includetissue and cell-specific promoters of an organism.

Gene: A DNA sequence that contains information needed for expressing apolypeptide or protein.

Structural gene: A DNA sequence that is transcribed into messenger RNA(mRNA) that is then translated into a sequence of amino acidscharacteristic of a specific polypeptide.

Complementary DNA (cDNA): A “complementary DNA,” or “cDNA” gene includesrecombinant genes synthesized by reverse transcription of mRNA and fromwhich intervening sequences (introns) have been removed.

Expression: Expression is the process by which a polypeptide is producedfrom a structural gene. The process involves transcription of the geneinto mRNA and the translation of such mRNA into polypeptide(s).

Homologous/Nonhomologous: Two nucleic acid molecules are considered tobe “homologous” if their nucleotide sequences share a similarity ofgreater than 40%, as determined by HASH-coding algorithms (Wilber, W. J.and Lipman, D. J., Proc. Natl. Acad. Sci. 80:726-730 (1983)). Twonucleic acid molecules are considered to be “nonhomologous” if theirnucleotide sequences share a similarity of less than 40%.

Polynucleotide: This term generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA. DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications have been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

Polypeptide: This term refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as post-translational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, Proteins-Structure and MolecularProperties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork. 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”. Methods in Enzymol. 182:626-646 (1990) and Rattan et al.“Protein Synthesis: Posttranslational Modifications and Aging”, Ann NYcad Sci 663:48-62 (1992).

Variant: The term used herein is a polynucleotide or polypeptide thatdiffers from a reference polynucleotide or polypeptide respectively, butretains essential properties. A typical variant of a polynucleotidediffers in nucleotide sequence from another, reference polynucleotide.Changes in the nucleotide sequence of the variant may or may not alterthe amino acid sequence of a polypeptide encoded by the referencepolynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

Identity: This term refers to a measure of the identity of nucleotidesequences or amino acid sequences. In general, the sequences are alignedso that the highest order match is obtained. “Identity” per se has anart-recognized meaning and can be calculated using published techniques.(See, e.g.: Computational Molecular Biology, Lesk, A. M. ed., OxfordUniversity Press, New York, 1988; Biocomputing, Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). While there exist a number of methods to measure identity betweentwo polynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math48:1073 (1988)). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H. & Lipton, D., SIAM J AppliedMath 48:1073 (1988). Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCS program package (Devereux, J. et al., Nucleic AcidsResearch 12(1):387(1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al.,J. Mol. Biol. 215:403 (1990)).

Therefore, as used herein, the term “identity” represents a comparisonbetween a test and reference polynucleotide. More specifically,reference polynucleotides are identified in this invention as SEQ IDNos: 1, 2, 3 and 4, and a test polynucleotide is defined as anypolynucleotide that is 90% or more identical to a referencepolynucleotide. As used herein, the term “90% or more” refers to percentidentities from 90 to 99.99 relative to the reference polynucleotide.Identity at a level of 90% or more is indicative of the fact that,assuming for exemplification purposes a test and referencepolynucleotide length of 100 nucleotides, that no more than 10% (i.e.,10 out of 100) nucleotides in the test polynucleotide differ from thatof the reference polynucleotide. Such differences may be represented aspoint mutations randomly distributed over the entire length of thesequence or they may be clustered in one or more locations of varyinglength up to the maximum allowable 10 nucleotide difference. Differencesare defined as nucleotide substitutions, deletions or additions ofsequence. These differences may be located at any position in thesequence, including but not limited to the 5′ end, 3′ end, coding andnon coding sequences.

Fragment: A “fragment” of a molecule such as de novo DNA cytosinemethyltransferases is meant to refer to any polypeptide subset of thatmolecule.

Functional Derivative: The term “functional derivatives” is intended toinclude the “variants,” “analogues,” or “chemical derivatives” of themolecule. A “variant” of a molecule such as de novo DNA cytosinemethyltransferases is meant to refer to a naturally occurring moleculesubstantially similar to either the entire molecule, or a fragmentthereof. An “analogue” of a molecule such as de novo DNA cytosinemethyltransferases is meant to refer to a non-natural moleculesubstantially similar to either the entire molecule or a fragmentthereof.

A molecule is said to be “substantially similar” to another molecule ifthe sequence of amino acids in both molecules is substantially the same,and if both molecules possess a similar biological activity. Thus,provided that two molecules possess a similar activity, they areconsidered variants as that term is used herein even if one of themolecules contains additional amino acid residues not found in theother, or if the sequence of amino acid residues is not identical.

As used herein, a molecule is said to be a “chemical derivative” ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties may improve themolecule's solubility, absorption, biological half-life, etc. Themoieties may alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, etc.Examples of moieties capable of mediating such effects are disclosed inRemington's Pharmaceutical Sciences (1980) and will be apparent to thoseof ordinary skill in the art.

Protein Activity or Biological Activity of the Protein: Theseexpressions refer to the metabolic or physiologic function of de novoDNA cytosine methyltransferase protein including similar activities orimproved activities or these activities with decreased undesirableside-effects. Also included are antigenic and immunogenic activities ofsaid de novo DNA cytosine methyltransferase protein. Among thephysiological or metabolic activities of said protein is the transfer ofa methyl group to the cytosine C5 position of duplex DNA. Such DNA maycompletely lack any methylation of may be hemimethylated. Asdemonstrated in Example 8, de novo DNA cytosine methyltransferasesmethylate C5 in cytosine moieties in nonmethylated DNA.

De novo DNA Cytosine Methyltransferases Polynucleotides: This termrefers to a polynucleotide containing a nucleotide sequence whichencodes a de novo DNA cytosine methyltransferase polypeptide or fragmentthereof or that encodes a de novo DNA cytosine methyltransferasepolypeptide or fragment wherein said nucleotide sequence has at least90% identity to a nucleotide sequence encoding the polypeptide of SEQ IDNos: 5, 6, 7 or 8, or a corresponding fragment thereof, or which hassufficient identity to a nucleotide sequence contained in SEQ ID NO:1,2, 3 or 4.

De novo DNA Cytosine Methyltransferases Polypeptides: This term refersto polypeptides with amino acid sequences sufficiently similar to the denovo DNA cytosine methyltransferase protein sequence in SEQ ID NO:5, 6,7 or 8 and that at least one biological activity of the protein isexhibited.

Antibodies: As used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

Substantially pure: As used herein means that the desired purifiedprotein is essentially free from contaminating cellular components, saidcomponents being associated with the desired protein in nature, asevidenced by a single band following polyacrylamide-sodium dodecylsulfate gel electrophoresis. Contaminating cellular components mayinclude, but are not limited to, proteinaceous, carbohydrate, or lipidimpurities.

The term “substantially pure” is further meant to describe a moleculewhich is homogeneous by one or more purity or homogeneitycharacteristics used by those of skill in the art. For example, asubstantially pure de novo DNA cytosine methyltransferases will showconstant and reproducible characteristics within standard experimentaldeviations for parameters such as the following: molecular weight,chromatographic migration, amino acid composition, amino acid sequence,blocked or unblocked N-terminus, HPLC elution profile, biologicalactivity, and other such parameters. The term, however, is not meant toexclude artificial or synthetic mixtures of the factor with othercompounds. In addition, the term is not meant to exclude de novo DNAcytosine methyltransferase fusion proteins isolated from a recombinanthost.

Isolated: A term meaning altered “by the hand of man” from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated”, as the term is employed herein. Thus, a polypeptide orpolynucleotide produced and/or contained within a recombinant host cellis considered isolated for purposes of the present invention. Alsointended as an “isolated polypeptide” or an “isolated polynucleotide”are polypeptides or polynucleotides that have been purified, partiallyor substantially, from a recombinant host cell or from a native source.For example, a recombinantly produced version of a de novo DNA cytosinemethyltransferase polypeptide can be substantially purified by theone-step method described in Smith and Johnson, Gene 67.31-40 (1988).

Neoplastic disorder: This term refers to a disease state which isrelated to the hyperproliferation of cells. Neoplastic disordersinclude, but are not limited to, carcinomas, sarcomas and leukemias.

Gene Therapy: A means of therapy directed to altering the normal patternof gene expression of an organism. Generally, a recombinantpolynucleotide is introduced into cells or tissues of the organism toeffect a change in gene expression.

Antisense RNA gene/Antisense RNA. In eukaryotes, mRNA is transcribed byRNA polymerase II. However, it is also known that one may construct agene containing a RNA polymerase II template wherein a RNA sequence istranscribed which has a sequence complementary to that of a specificmRNA but is not normally translated. Such a gene construct is hereintermed an “antisense RNA gene” and such a RNA transcript is termed an“antisense RNA.” Antisense RNAs are not normally translatable due to thepresence of translation stop codons in the antisense RNA sequence.

Antisense oligonucleotide: A DNA or RNA molecule or a derivative of aDNA or RNA molecule containing a nucleotide sequence which iscomplementary to that of a specific mRNA. An antisense oligonucleotidebinds to the complementary sequence in a specific mRNA and inhibitstranslation of the mRNA. There are many known derivatives of such DNAand RNA molecules. See, for example, U.S. Pat. Nos. 5,602,240,5,596,091, 5,506,212, 5,521,302, 5,541,307, 5,510,476, 5,514,787,5,543,507, 5,512,438, 5,510,239, 5,514,577, 5,519,134, 5,554,746,5,276,019, 5,286,717, 5,264,423, as well as WO96/35706, WO96/32474,WO96/29337 (thiono triester modified antisense oligodeoxynucleotidephosphorothioates), WO94/17093 (oligonucleotide alkylphosphonates andalkylphosphothioates), WO94/08004 (oligonucleotide phosphothioates,methyl phosphates, phosphoramidates, dithioates, bridgedphosphorothioates, bridge phosphoramidates, sulfones, sulfates, ketos,phosphate esters and phosphorobutylamines (van der Krol et al., Biotech.6:958-976 (1988); Uhlmann et al., Chem. Rev. 90:542-585 (1990)),WO94/02499 (oligonucleotide alkylphosphonothioates andarylphosphonothioates), and WO92/20697 (3′-end capped oligonucleotides).Particular de novo DNA cytosine methyltransferase antisenseoligonucleotides of the present invention include derivatives such asS-oligonucleotides (phosphorothioate derivatives or S-oligos, see, JackCohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression,CRC Press (1989)). S-oligos (nucleoside phosphorothioates) areisoelectronic analogs of an oligonucleotide (O-oligo) in which anonbridging oxygen atom of the phosphate group is replaced by a sulfuratom. The S-oligos of the present invention may be prepared by treatmentof the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxidewhich is a sulfur transfer reagent. See Iyer et al., J. Org. Chem.55:4693-4698 (1990); and Iyer et al., J. Am. Chem. Soc. 112:1253-1254(1990).

Antisense Therapy: A method of treatment wherein antisenseoligonucleotides are administered to a patient in order to inhibit theexpression of the corresponding protein.

I. Deposited Material

The invention relates to polynucleotides encoding and polypeptides ofnovel de novo DNA cytosine methyltransferase proteins. The inventionrelates especially to de novo DNA cytosine methyltransferase mouseDnmt3a and Dnmt3b cDNAs and the human DNMT3A and DNMT3B cDNAs set out inSEQ ID NOs: 1, 2, 3 and 4, respectively. The invention also relates tomouse Dnmt3a and Dnmt3b nd human DNMT3A and DNMTB e novo DNA cytosinemethyltransferase polypeptides set out in SEQ ID NOs:5, 6, 7, and 8,respectively. The invention further relates to the de novo DNA cytosinemethyltransferase nucleotide sequences of the mouse Dnmt3a cDNA (plasmidpMT3a) and Dnmt3b cDNA (plasmid pMT3b) and the human DNMTα cDNA (plasmidpMT3A) in ATCC Deposit Nos. 209933, 209934, and 98809, respectively, andthe amino acid sequences encoded therein.

The nucleotide sequence of the human DNMT3B cDNA identified in SEQ IDNO:4 is available in a clone (ATCC Deposit No. 326637) independentlydeposited by the I.M.A.G.E. Consortium. The invention relates to the denovo DNA cytosine methyltransferase polypeptide encoded therein.

Clones containing mouse Dnmt3a and Dnmt3b cDNAs were deposited with theAmerican Type Culture Collection (ATCC), Manassas, Va. 20110-2209, USA,on Jun. 16, 1998, and assigned ATCC Deposit Nos. 209933 and 209934,respectively. The human DNMT3A cDNA was deposited with the ATCC on Jul.10, 1998, and assigned ATCC Deposit No. 98809.

While the ATCC deposits are believed to contain the de novo DNA cytosinemethyltransferase cDNA sequences shown in SEQ ID NOs:1, 2, 3, and 4, thenucleotide sequences of the polynucleotide contained in the depositedmaterial, as well as the amino acid sequence of the polypeptide encodedthereby, are controlling in the event of any conflict with anydescription of sequences herein.

The deposits for mouse Dnmt3a and Dnmt3b cDNAs and the human DNMT3A cDNAwere made under the terms of the Budapest Treaty on the internationalrecognition of the deposit of micro-organisms for purposes of patentprocedure. The deposits are provided merely as a convenience for thoseof skill in the art and are not an admission that a deposit is requiredfor enablement, such as that required under 35 U.S.C. § 112.

II. Polynucleotides of the Invention

Another aspect of the invention relates to isolated polynucleotides, andpolynucleotides closely related thereto, which encode the de novo DNAcytosine methyltransferase polypeptides. As shown by the resultspresented in FIG. 5, sequencing of the cDNAs contained in the depositedclones encoding mouse and human de novo DNA cytosine methyltransferasesconfirms that the de novo DNA cytosine methyltransferase proteins of theinvention are structurally related to other proteins of the DNAmethyltransferase family.

The polynucleotides of the present invention encoding de novo DNAcytosine methyltransferase proteins may be obtained using standardcloning and screening procedures as described in Example 1.Polynucleotides of the invention can also be obtained from naturalsources such as genomic DNA libraries or can be synthesized using wellknown and commercially available techniques.

Among particularly preferred embodiments of the invention arepolynucleotides encoding de novo DNA cytosine methyltransferasepolypeptides having the amino acid sequence set out in SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, or SEQ ID NO:8, and variants thereof.

A particular nucleotide sequence encoding a de novo DNA cytosinemethyltransferase polypeptide may be identical over its entire length tothe coding sequence in SEQ ID NOs:1, 2, or 3. Alternatively, aparticular nucleotide sequence encoding a de novo DNA cytosinemethyltransferase polypeptide may be an alternate form of SEQ ID NOs:1,2, 3 and 4 due to degeneracy in the genetic code or variation in codonusage encoding the polypeptides of SEQ ID NOs:5, 6, 7, or 8. Preferably,the polynucleotides of the invention contain a nucleotide sequence thatis highly identical, at least 90% identical, with a nucleotide sequenceencoding a de novo DNA cytosine methyltransferase polypeptide or atleast 90% identical with the encoding nucleotide sequence set forth inSEQ ID NOs:1, 2, or 3. Polynucleotides of the invention may be 90 to 99%identical to the nucleotides sequence set forth in SEQ ID NO:4.

When a polynucleotide of the invention is used for the recombinantproduction of a de novo DNA cytosine methyltransferase polypeptide, thepolynucleotide may include the coding sequence for the full-lengthpolypeptide or a fragment thereof, by itself; the coding sequence forthe full-length polypeptide or fragment in reading frame with othercoding sequences, such as those encoding a leader or secretory sequence,a pre-, or pro or prepro-protein sequence, or other fusion peptideportions. For example, a marker sequence that facilitates purificationof the fused polypeptide can be encoded. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al, Proc Natl Acad Sci USA 86:821-824 (1989), orit may be the HA tag, which corresponds to an epitope derived from theinfluenza hemagglutinin protein (Wilson, I., et al, Cell 37:767, 1984).The polynucleotide may also contain non-coding 5′ and 3′ sequences, suchas transcribed, non-translated sequences, splicing and polyadenylationsignals, ribosome binding sites and sequences that stabilize mRNA.

Embodiments of the invention include isolated nucleic acid moleculescomprising a polynucleotide having a nucleotide sequence at least 90%identical, and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to (a) a nucleotide sequence encoding a denovo DNA cytosine methyltransferase polypeptide having the amino acidsequence in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; (b) a nucleotidesequence encoding a de novo DNA cytosine methyltransferase polypeptidehaving the amino acid sequence encoded by the cDNA clone contained inATCC Deposit No. 209933, ATCC Deposit No. 209934, or ATCC Deposit No.98809; or (c) a nucleotide sequence complementary to any of thenucleotide sequences in (a) or (b). Additionally, an isolated nucleicacid of the invention may be a polynucleotide at least 90% but not morethan 99% identical to (a) a nucleotide sequence encoding a de novo DNAcytosine methyltransferase polypeptide having the amino acid sequence inSEQ ID NO:4; (b) a nucleotide sequence encoding a de novo DNA cytosinemethyltransferase polypeptide having the amino acid sequence encoded bythe cDNA clone contained in ATCC Deposit No. 326637; or (c) a nucleotidesequence complementary to any of the nucleotide sequences in (a) or (b).

Conventional means utilizing known computer programs such as the BestFitprogram (Wisconsin Sequence Analysis Package, Version 10 for Unix,Genetics Computer Group. University Research Park, 575 Science Drive,Madison, Wis. 53711) may be utilized to determine if a particularnucleic acid molecule is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to any one of the nucleotide sequences shownin SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 or to any oneof the nucleotide sequences of the deposited cDNA clones contained inATCC Deposit No. 209933, ATCC Deposit No. 209934, ATCC Deposit No.98809, or ATCC Deposit No. 326637.

Further preferred embodiments are polynucleotides encoding de novo DNAcytosine methyltransferases and de novo DNA cytosine methyltransferasevariants that have an amino acid sequence of the de novo DNA cytosinemethyltransferase protein of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, orSEQ ID NO:8 in which several, 1, 1-2, 1-3, 1-5 or 5-10 amino acidresidues are substituted, deleted or added, in any combination.

Further preferred embodiments of the invention are polynucleotides thatare at least 90% identical over their entire length to a polynucleotideencoding a de novo DNA cytosine methyltransferase polypeptide having theamino acid sequence set out in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, orSEQ ID NO:8, and polynucleotides which are complementary to suchpolynucleotides. Most highly preferred are polynucleotides that compriseregions that are at least 90% identical over their entire length to apolynucleotide encoding the de novo DNA cytosine methyltransferasepolypeptides of the ATCC deposited human DNMT3A cDNA clone andpolynucleotides complementary thereto, and 90% to 99% identical overtheir entire length to a polynucleotide encoding the de novo DNAcytosine methyltransferase polypeptides of the ATCC deposited humanDNMT3B cDNA clone and polynucleotides complementary thereto. In thisregard, polynucleotides at least 95% identical over their entire lengthto the same are particularly preferred, and those with at least 97%identity are especially preferred. Furthermore, those with at least 98%identity are highly preferred and with at least 99% identity being themost preferred.

In a more specific embodiment, the nucleic acid molecules of the presentinvention, e.g. isolated nucleic acids comprising a polynucleotidehaving a nucleotide sequence encoding a de novo DNA cytosinemethyltransferase polypeptide or fragment thereof, are not the sequenceof nucleotides, the nucleic acid molecules (e.g., clones), or thenucleic acid inserts identified in one or more of the below cited publicEST or STS GenBank Accession Reports.

The following public ESTs were identified that relate to portions of SEQID NO:1: AA052791(SEQ ID NO:9); AA111043(SEQ ID NO:10); AA154890(SEQ IDNO:11); AA240794(SEQ ID NO:12); AA756653(SEQ ID NO:13); W58898(SEQ IDNO:14); W59299(SEQ ID NO:15); W91664(SEQ ID NO:16); W91665(SEQ IDNO:17); to portions of SEQ ID NO:2: AA116694 (SEQ ID NO:18); AA119979(SEQ ID NO:19); AA177277 (SEQ ID NO:20); AA210568 (SEQ ID NO:21);AA399749 (SEQ ID NO:22); AA407106 (SEQ ID NO:23); AA575617 (SEQ IDNO:24); to portions of SEQ ID NO:3: AA004310 (SEQ ID NO:25); AA004399(SEQ ID NO:26); AA312013 (SEQ ID NO:27); AA355824 (SEQ ID NO:28);AA533619 (SEQ ID NO:29); AA361360 (SEQ ID NO:30); AA364876 (SEQ IDNO:31); AA503090 (SEQ ID NO:32); AA533619 (SEQ ID NO:33); AA706672 (SEQID NO:34); AA774277 (SEQ ID NO:35); AA780277 (SEQ ID NO:36); H03349 (SEQID NO:37); H04031 (SEQ ID NO:38); H53133 (SEQ ID NO:39); H53239 (SEQ IDNO:40); H64669 (SEQ ID NO:41); N26002 (SEQ ID NO:42); N52936 (SEQ IDNO:43); N88352 (SEQ ID NO:44); N89594 (SEQ ID NO:45); R19795 (SEQ IDNO:46); R47511 (SEQ ID NO:47); T50235 (SEQ ID NO:48); T78023 (SEQ IDNO:49); T78186 (SEQ ID NO:50); W22886 (SEQ ID NO:51); W67657 (SEQ IDNO:52); W68094 (SEQ ID NO:53); W76111 (SEQ ID NO:54); Z38299 (SEQ IDNO:55); Z42012 (SEQ ID NO:56); and that relate to SEQ ID NO:4:AA206103(SEQ ID NO:57); AA206264(SEQ ID NO:58); AA216527(SEQ ID NO:59);AA216697(SEQ ID NO:60); AA305044(SEQ ID NO:61); AA477705(SEQ ID NO:62);AA477706(SEQ ID NO:63); AA565566(SEQ ID NO:64); AA599893(SEQ ID NO:65);AA729418(SEQ ID NO:66); AA887508(SEQ ID NO:67); F09856(SEQ ID NO:68);F12227(SEQ ID NO:69); N39452(SEQ ID NO:70); N48564(SEQ ID NO:71);T66304(SEQ ID NO:72); and T66356(SEQ ID NO:73); AA736582(SEQ ID NO:77);AA748883(SEQ ID NO:78); AA923295(SEQ ID NO:79); AA1000396(SEQ ID NO:80);A1332472(SEQ ID NO:81); W22473(SEQ ID NO:82) and the I.M.A.G.E.Consortium clone ID 22089 (ATCC Deposit No. 326637)(SEQ ID NO:76).Additionally, STSs G06200(SEQ ID NO:74) and G15302(SEQ ID NO:75) wereidentified in a search with SEQ ID NOS.:3 and 4, respectively.

The present invention is further directed to fragments of SEQ ID NO:1, 2or 3, or to fragments of the cDNA nucleotide sequence found in ATCCDeposit Nos. 209933, 209934, or 98809. A fragment may be defined to beat least about 15 nt, and more preferably at least about 20 nt, stillmore preferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length. Such fragments are useful as diagnostic probesand primers as discussed herein. Of course larger DNA fragments are alsouseful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thecDNA clones contained in the plasmids deposited as ATCC Deposit No.209933, ATCC Deposit No. 209934 or ATCC Deposit No. 98809; or as shownin SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. Generally, polynucleotidefragments of the invention may be defined algebraically in the followingway: (a) for SEQ ID NO:1, as 15+N, wherein N equals zero or any positiveinteger up to 4176; (b) for SEQ ID NO:2, as 15+N, wherein N equals zeroor any positive integer up to 4180; and (c) for SEQ ID NO:3, as 15+N,wherein N equals zero or any positive integer up to 4401. By a fragmentat least 20 nt in length, for example, is intended fragments whichinclude 20 or more contiguous bases from a nucleotide sequence of theATCC deposited cDNAs or the nucleotide sequence as shown in SEQ ID NO:1,SEQ ID NO:2 or SEQ ID NO:3.

In another embodiment, the invention is directed to fragments of SEQ IDNO:4. Such fragments are defined as comprising the nucleotide sequenceencoding the specific amino acid residues integral and immediatelyadjacent to the site where DNMT3B exons are spliced together. The DNMT3Bsequence of SEQ ID NO:4 consists of 23 exon sequences definedaccordingly: Exon 1 consists of nucleotides 1-108 of SEQ ID NO:4; Exon 2consists of nucleotides 109-256 of SEQ ID NO:4; Exon 3 consists ofnucleotides 257-318 of SEQ ID NO:4; Exon 4 consists of nucleotides319-420 of SEQ ID NO:4; Exon 5 consists of nucleotides 421-546 of SEQ IDNO:4; Exon 6 consists of nucleotides 547-768 of SEQ ID NO:4; Exon 7consists of nucleotides 769-927 of SEQ ID NO:4; Exon 8 consists ofnucleotides 928-1035 of SEQ ID NO:4; Exon 9 consists of nucleotides1036-1180 of SEQ ID NO:4; Exon 10 consists of nucleotides 1181-1240 ofSEQ ID NO:4; Exon 11 consists of nucleotides 1241-1366 of SEQ ID NO:4;Exon 12 consists of nucleotides 1367-1411 of SEQ ID NO:4; Exon 13consists of nucleotide 1412-1491 of SEQ ID NO:4. Exon 14 consists ofnucleotides 1492-1604 of SEQ ID NO:4; Exon 15 consists of nucleotides1605-1788 of SEQ ID NO:4; Exon 16 consists of nucleotides 1789-1873 ofSEQ ID NO:4; Exon 17 consists of nucleotides 1874-2019 of SEQ ID NO:4;Exon 18 consists of nucleotides 2020-2110 of SEQ ID NO:4; Exon 19consists of nucleotides 2111-2259 of SEQ ID NO:4; Exon 20 consists ofnucleotides 2260-2345 of SEQ ID NO:4; Exon 21 consists of nucleotides2346-2415 of SEQ ID NO:4; Exon 22 consists of nucleotides 2416-2534 ofSEQ ID NO:4; and Exon 23 consists of nucleotides 2535-4145 of SEQ IDNO:4.

It should be understood by those skilled in the art that with regards toSEQ ID NO:4. Exon 1 and Exon 23 are herein defined for the purposes ofthe invention. The first nucleotide of Exon 1 may or may not be thetranscriptional start site for the DNMT3B genomic locus, and the lastnucleotide identified for Exon 23 may or may not reflect the lastnucleotide transcribed in vivo.

Thus, by way of example, fragments of SEQ ID NO:4 comprise the followingexon-exon junctions of 20 nucleotides in length: the exon1/exon 2junction of nucleotides 98-118 of SEQ ID NO:4; the exon 2/exon 3junction of nucleotides 246-266 of SEQ ID NO:4; the exon 3/exon 4junction of nucleotides 308-328 of SEQ ID NO:4; the exon 4/exon 5junction of nucleotides 410-430 of SEQ ID NO:4; the exon 5/exon 6junction of nucleotides 536-556 of SEQ ID NO:4; the exon 6/exon 7junction of nucleotides 758-778 of SEQ ID NO:4; the exon 7/exon 8junction of nucleotides 917-937 of SEQ ID NO:4; the exon 8/exon 9junction of nucleotides 1025-1045 of SEQ ID NO:4; the exon 9/exon 10junction of nucleotides 1170-1190 of SEQ ID NO:4; the exon 10/exon 11junction of nucleotides 1230-1250 of SEQ ID NO:4; the exon 11/exon 12junction of nucleotides 1356-1376 of SEQ ID NO:4; the exon 12/exon 13junction of nucleotides 1401-1421 of SEQ ID NO:4; the exon 13/exon 14junction of nucleotides 1481-1501 of SEQ ID NO:4; the exon 14/exon 15junction of nucleotides 1594-1614 of SEQ ID NO:4; the exon 15/exon 16junction of nucleotides 1778-1798 of SEQ ID NO:4; the exon 16/exon 17junction of nucleotides 1863-1883 of SEQ ID NO:4; the exon 17/exon 18junction of nucleotides 2009-2029 of SEQ ID NO:4; the exon 18/exon 19junction of nucleotides 2100-2120 of SEQ ID NO:4; the exon 19/exon 20junction of nucleotides 2249-2269 of SEQ ID NO:4; the exon 20/exon 21junction of nucleotides 2335-2355 of SEQ ID NO:4; the exon 21/exon 22junction of nucleotides 2405-2425 of SEQ ID NO:4; and the exon 22/exon23 junction of nucleotides 2524-2544 of SEQ ID NO:4.

As will be clear to those skilled in the art, other exon-exon junctionfragments of SEQ ID NO:4 are possible which comprise 30, 40, 50, 60, 70,80, 90, 100, 200, 300, 400, 500, etc., nucleotides of SEQ ID NO:4. Forthe purposes of constructing such fragments, the followingexon-exon/junctions are identified: the exon1/exon 2 junction ofnucleotides 108 and 109 of SEQ ID NO:4; the exon 2/exon 3 junction ofnucleotides 256 and 257 of SEQ ID NO:4; the exon 3/exon 4 junction ofnucleotides 318 and 319 of SEQ ID NO:4; the exon 4/exon 5 junction ofnucleotides 420 and 421 of SEQ ID NO:4; the exon 5/exon 6 junction ofnucleotides 546 and 547 of SEQ ID NO:4; the exon 6/exon 7 junction ofnucleotides 768 and 769 of SEQ ID NO:4; the exon 7/exon 8 junction ofnucleotides 927 and 928 of SEQ ID NO:4; the exon 8/exon 9 junction ofnucleotides 1035 and 1036 of SEQ ID NO:4; the exon 9/exon 10 junction ofnucleotides 1180 and 1181 of SEQ ID NO:4; the exon 10/exon 11 junctionof nucleotides 1240 and 1241 of SEQ ID NO:4: the exon 11/exon 12junction of nucleotides 1366 and 1367 of SEQ ID NO:4; the exon 12/exon13 junction of nucleotides 1411 and 1412 of SEQ ID NO:4; the exon13/exon 14 junction of nucleotides 1491 and 1492 of SEQ ID NO:4; theexon 14/exon 15 junction of nucleotides 1604 and 1605 of SEQ ID NO:4;the exon 15/exon 16 junction of nucleotides 1788 and 1789 of SEQ IDNO:4; the exon 16/exon 17 junction of nucleotides 1873 and 1874 of SEQID NO:4; the exon 17/exon 18 junction of nucleotides 2019 and 2020 ofSEQ ID NO:4; the exon 18/exon 19 junction of nucleotides 2110 and 2111of SEQ ID NO:4; the exon 19/exon 20 junction of nucleotides 2259 and2260 of SEQ ID NO:4; the exon 20/exon 21 junction of nucleotides 2345and 2346 of SEQ ID NO:4; the exon 20/exon 22 junction of nucleotides2415 and 2416 of SEQ ID NO:4; and the exon 22/exon 23 junction ofnucleotides 2534 and 2535 of SEQ ID NO:4. Junction nucleotides may belocated at any position of the selected SEQ ID NO:4 fragment.

The present invention further relates to polynucleotides that hybridizeto the above-described sequences. In this regard, the present inventionespecially relates to polynucleotides that hybridize under stringentconditions to the above-described polynucleotides. As herein used, theterm “stringent conditions” means hybridization will occur only if thereis at least 90% and preferably at least 95% identity and more preferablyat least 97% identity between the sequences.

Furthermore, a major consideration associated with hybridizationanalysis of DNA or RNA sequences is the degree of relatedness the probehas with the sequences present in the specimen under study. This isimportant with a blotting technique (e.g., Southern or Northern Blot),since a moderate degree of sequence homology under nonstringentconditions of hybridization can yield a strong signal even though theprobe and sequences in the sample represent non-homologous genes.

The particular hybridization technique is not essential to theinvention, any technique commonly used in the art is within the scope ofthe present invention. Typical probe technology is described in U.S.Pat. No. 4,358,535 to Falkow et al., incorporated by reference herein.For example, hybridization can be carried out in a solution containing6×SSC (10×SSC: 1.5 M sodium chloride, 0.15 M sodium citrate, pH 7.0), 5×Denhardt's (1× Denhardt's: 0.2% bovine serum albumin, 0.2%polyvinylpyrrolidone, 0.02% Ficoll 400), 10 mM EDTA, 0.5% SDS and about10⁷ cpm of nick-translated DNA for 16 hours at 65° C. Additionally, ifhybridization is to an immobilized nucleic acid, a washing step may beutilized wherein probe binding to polynucleotides of low homology, ornonspecific binding of the probe, may be removed. For example, astringent wash step may involve a buffer of 0.2×SSC and 0.5% SDS at atemperature of 65° C.

Additional information related to hybridization technology and, moreparticularly, the stringency of hybridization and washing conditions maybe found in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1989), which is incorporated herein by reference.

Polynucleotides of the invention which are sufficiently identical to anucleotide sequences contained in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3or SEQ ID NO:4, or in the cDNA inserts of ATCC Deposit No. 209933, ATCCDeposit No. 209934, ATCC Deposit No. 98809 or ATCC Deposit No. 326637,may be used as hybridization probes for cDNA and genomic DNA, to isolatefull-length cDNAs and genomic clones encoding de novo DNA cytosinemethyltransferase proteins and to isolate cDNA and genomic clones ofother genes that have a high sequence similarity to the de novo DNAcytosine methyltransferase genes. Such hybridization techniques areknown to those of skill in the art. Typically, these nucleotidesequences are at least about 90% identical, preferably at least about95% identical, more preferably at least about 97%, 98% or 99% identicalto that of the reference. The probes generally will comprise at least 15nucleotides. Preferably, such probes will have at least 30 nucleotidesand may have at least 50 nucleotides. Particularly preferred probes willrange between 30 and 50 nucleotides.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to animal and human disease.

III. Vectors, Host Cells, and Recombinant Expression

The present invention also relates to vectors that comprise apolynucleotide of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems for polynucleotides of the invention.Introduction of polynucleotides into host cells can be effected bymethods described in many standard laboratory manuals, such as Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). For example,calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introduction,infection or any other means known in the art may be utilized.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci. E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insert cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episomal and virus-derived systems, e.g.,vectors derived from bacterial plasmids, from bacteriophages, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses, and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., Molecular Cloning: A Laboratory Manual (supra).

RNA vectors may also be utilized for the expression of the de novo DNAcytosine methyltransferases disclosed in this invention. These vectorsare based on positive or negative strand RNA viruses that naturallyreplicate in a wide variety of eukaryotic cells (Bredenbeek, P. J. andRice, C. M., Virology 3: 297-310, (1992)). Unlike retroviruses, theseviruses lack an intermediate DNA life-cycle phase, existing entirely inRNA form. For example, alpha viruses are used as expression vectors forforeign proteins because they can be utilized in a broad range of hostcells and provide a high level of expression; examples of viruses ofthis type include the Sindbis virus and Semliki Forest virus(Schlesinger, S., TIBTECH 11: 18-22, (1993); Frolov, I., et al., Proc.Natl. Acad. Sci. (USA) 93: 11371-11377, (1996)). As exemplified byInvitrogen's Sinbis expression system, the investigator may convenientlymaintain the recombinant molecule in DNA form (pSinrep5 plasmid) in thelaboratory, but propagation in RNA form is feasible as well. In the hostcell used for expression, the vector containing the gene of interestexists completely in RNA form and may be continuously propagated in thatstate if desired.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

As used herein, the term “operably linked,” when used in the context ofa linkage between a structural gene and an expression control sequence,e.g., a promoter, refers to the position and orientation of theexpression control sequence relative to the structural gene so as topermit expression of the structural gene in any host cell. For example,an operable linkage would maintain proper reading frame and would notintroduce any in frame stop codons.

As used herein, the term “heterologous promoter,” refers to a promoternot normally and naturally associated with the structural gene to beexpressed. For example, in the context of expression of a de novo DNAcytosine methyltransferase polypeptide, a heterologous promoter would beany promoter other than an endogenous promoter associated with the denovo DNA cytosine methyltransferase gene in non-recombinant mouse orhuman chromosomes. In specific embodiments of this invention, theheterologous promoter is a prokaryotic or bacteriophage promoter, suchas the lac promoter, T3 promoter, or T7 promoter. In other embodiments,the heterologous promoter is a eukaryotic promoter.

In other embodiments, this invention provides an isolated nucleic acidmolecule comprising a de novo DNA cytosine methyltransferase structuralgene operably linked to a heterologous promoter. As used herein, theterm “a de novo DNA cytosine methyltransferase structural gene” refersto a nucleotide sequence at least about 90% identical to one of thefollowing nucleotide sequences:

(a) a nucleotide sequence encoding the de novo DNA cytosinemethyltransferase polypeptide having the complete amino acid sequence inSEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7;

(b) a nucleotide sequence encoding the de novo DNA cytosinemethyltransferase polypeptide having the complete amino acid sequenceencoded by the cDNA insert of ATCC Deposit No. 209933, ATCC Deposit No.209934, or ATCC Deposit No. 98809; or

(c) a nucleotide sequence complementary to any of the nucleotidesequences in (a) or (b).

In preferred embodiments, the de novo DNA cytosine methyltransferasestructural gene is 90%, and more preferably 91%, 92%, 93%, 94%, 95%,97%, 98%, 99%, or 100% identical to one or more of nucleotide sequences(a), (b), or (c) supra.

In another embodiment the term “a de novo DNA cytosine methyltransferasestructural gene” refers to a nucleotide sequence about 90% to 99%identical to one of the following nucleotide sequences:

(a) a nucleotide sequence encoding the de novo DNA cytosinemethyltransferase polypeptide having the complete amino acid sequence inSEQ ID NO:8:

(b) a nucleotide sequence encoding the de novo DNA cytosinemethyltransferase polypeptide having the complete amino acid sequenceencoded by the cDNA insert of ATCC Deposit No. 326637; or

(c) a nucleotide sequence complementary to any of the nucleotidesequences in (a) or (b).

In preferred embodiments, the de novo DNA cytosine methyltransferasestructural gene is 90%, and more preferably 91%, 92%, 93%, 94%, 95%,97%, 98%, or 99% identical to SEQ ID NO:8, ATCC Deposit No. 326637 orpolynucleotides complementary thereto.

This invention also provides an isolated nucleic acid moleculecomprising a de novo DNA cytosine methyltransferase structural geneoperably linked to a heterologous promoter, wherein said isolatednucleic acid molecule does not encode a fusion protein comprising the denovo DNA cytosine methyltransferase structural gene or a fragmentthereof.

This invention further provides an isolated nucleic acid moleculecomprising a de novo DNA cytosine methyltransferase structural geneoperably linked to a heterologous promoter, wherein said isolatednucleic acid molecule is capable of expressing a de novo DNA cytosinemethyltransferase polypeptide when used to transform an appropriate hostcell.

This invention also provides an isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to asequence encoding a de novo DNA cytosine methyltransferase polypeptidehaving the amino acid sequence of SEQ ID NO:5. SEQ ID NO:6, SEQ ID NO:7or SEQ ID NO:8, wherein said isolated nucleic acid molecule does notcontain a nucleotide sequence at least 90% identical to the 3′untranslated region of SEQ ID NO:1 (nucleotides 2942-4191), SEQ ID NO:2(nucleotides 2847-4174), SEQ ID NO:3 (nucleotides 3090-4397) or SEQ IDNO:4 (nucleotides 2677-4127), or a fragment of the 3′ untranslatedregion greater than 25, 50, 75, 100, or 125 bp in length.

This invention further provides an isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to asequence encoding a de novo DNA cytosine methyltransferase polypeptidehaving the amino acid sequence of SEQ ID NO:5. SEQ ID NO:6, SEQ ID NO:7or SEQ ID NO:8, wherein said isolated nucleic acid molecule does notcontain a nucleotide sequence at least 90% identical to the 5′untranslated region of SEQ ID NO:1 (nucleotides 1-216), SEQ ID NO:2(nucleotides 1-268), SEQ ID NO:3 (nucleotides 1-352) or SEQ ID NO:4(nucleotides 1-114), or a fragment of the 5′ untranslated region greaterthan 25, 35, 45, 55, 65, 75, 85, or 90 bp.

Suitable known prokaryotic promoters for use in the production ofproteins of the present invention include the E. coli lacI and lacZpromoters, the T3 and T7 promoters, the gpt promoter, the lambda PR andPL promoters and the trp promoter. Suitable eukaryotic promoters includethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous Sarcoma Virus (RSV), adenovirus promoter, Herpes viruspromoter, and metallothionein promoters, such as the mousemetallothionein-I promoter and tissue and organ-specific promoters knownin the art.

If the de novo DNA cytosine methyltransferase polypeptide is to beexpressed for use in screening assays, generally, it is preferred thatthe polypeptide be produced at the surface of the cell. In this event,the cells may be harvested prior to use in the screening assay. If denovo DNA cytosine methyltransferase polypeptide is secreted into themedium, the medium can be recovered in order to recover and purify thepolypeptide; if produced intracellularly, the cells must first be lysedbefore the polypeptide is recovered.

De novo DNA cytosine methyltransferase polypeptides can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography is employed forpurification. Well known techniques for refolding proteins may beemployed to regenerate active conformation when the polypeptide isdenatured during isolation and or purification.

IV. Polypeptides of the Invention

The de novo DNA cytosine methyltransferase polypeptides of the presentinvention include the polypeptide of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7 or SEQ ID NO:8, as well as polypeptides and fragments which haveactivity and have at least 90% identity to the polypeptide of SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or the relevant portionand more preferably at least 96%, 97% or 98% identity to the polypeptideof SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and still morepreferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to the polypeptide of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 orSEQ ID NO:8.

The polypeptides of the present invention are preferably provided in anisolated form.

The polypeptides of the present invention include the polypeptideencoded by the deposited cDNAs; a polypeptide comprising amino acidsfrom about 1 to about 908 in SEQ ID NO:5; a polypeptide comprising aminoacids from about 1 to about 859 in SEQ ID NO:6; a polypeptide comprisingamino acids from about 1 to about 912 in SEQ ID NO:7 and a polypeptidecomprising amino acids from about 1 to about 853 in SEQ ID NO:8; as wellas polypeptides which are at least about 90% identical, and morepreferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the polypeptides described above and also includeportions of such polypeptides with at least 30 amino acids and morepreferably at least 50 amino acids.

Polypeptides of the invention also include alternative splicing variantsof the Dnmt3 sequences disclosed herein. For example, alternativevariant spliced proteins of mouse Dnmt3b include but are not limited toa polypeptide wherein, except for at least one conservative amino acidsubstitution, said polypeptide has a sequence selected from the groupconsisting of: (1) amino acid residues 1 to 362 and 383 to 859 from SEQID NO:2; and (2) amino acid residues 1 to 362 and 383 to 749 and 813 to859 from SEQ ID NO:2; and alternative variant spliced proteins of humanDNMT3B include but are not limited to a polypeptide wherein, except forat least one conservative amino acid substitution, said polypeptide hasa sequence selected from the group consisting of: (1) amino acidresidues 1 to 355 and 376 to 853 from SEQ ID NO:4; and (2) amino acidresidues 1 to 355 and 376 to 743 and 807 to 853 from SEQ ID NO:4.

The de novo DNA cytosine methyltransferase polypeptides may be a part ofa larger protein such as a fusion protein. It is often advantageous toinclude additional amino acid sequence which contains secretory orleader sequences, pro-sequences, sequences which aid in purificationsuch as multiple histidine residues, or additional sequence forstability during recombinant production.

Biologically active fragments of the de novo DNA cytosinemethyltransferase polypeptides are also included in the invention. Afragment is a polypeptide having an amino acid sequence that entirely isthe same as part but not all of the amino acid sequence of one of theaforementioned de novo DNA cytosine methyltransferase polypeptides. Aswith de novo DNA cytosine methyltransferase polypeptides, fragments maybe “free-standing,” or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion. In the context of this invention, a fragment may constitute fromabout 10 contiguous amino acids identified in SEQ ID NO:5, SEQ ID NO:6.SEQ ID NO:7 or SEQ ID NO:8. More specifically, polypeptide fragmentlengths may be defined algebraically as follows: (a) for SEQ ID NO:5, as10+N, wherein N equals zero or any positive integer up to 898; (b) forSEQ ID NO:6, as 10+N, wherein N equals zero or any positive integer upto 849; (c) for SEQ ID NO:7, as 10+N, wherein N equals zero or anypositive integer up to 902; and (d) for SEQ ID NO:8, as 10+N, wherein Nequals zero or any positive integer up to 843.

Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of de novo DNA cytosine methyltransferasepolypeptides, except for deletion of a continuous series of residuesthat includes the amino terminus, or a continuous series of residuesthat includes the carboxyl terminus or deletion of two continuous seriesof residues, one including the amino terminus and one including thecarboxyl terminus. Also preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Biologically active fragments are those that mediateprotein activity, including those with a similar activity or an improvedactivity, or with a decreased undesirable activity. Also included arethose that are antigenic or immunogenic in an animal, especially in ahuman.

Thus, the polypeptides of the invention include polypeptides having anamino acid sequence at least 90% identical to that of SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or fragments thereof with at least90% identity to the corresponding fragment of SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7 or SEQ ID NO:8, all of which retain the biological activityof the de novo DNA cytosine methyltransferase protein, includingantigenic activity. Included in this group are variants of the definedsequence and fragment. Preferred variants are those that vary from thereference by conservative amino acid substitutions, i.e. those thatsubstitute a residue with another of like characteristics. Typicalsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg, or aromatic residues Phe and Tyr. Particularlypreferred are variants in which several, 5 to 10, 1 to 5, or 1 to 2amino acids are substituted, deleted, or added in any combination.

The de novo DNA cytosine methyltransferase polypeptides of the inventioncan be prepared in any suitable manner. Such polypeptides includeisolated naturally occurring polypeptides, recombinantly producedpolypeptides, synthetically produced polypeptides, or polypeptidesproduced by a combination of these methods. Means for preparing suchpolypeptides are well understood in the art.

V. In Vitro DNA Methylation

One preferred embodiment of the invention enables the in vitromethylation at the C5 position of cytosine in DNA. The startingsubstrate DNA may be hemimethylated (i.e., one strand of the duplex DNAis methylated) or may lack methylation completely. The polypeptides ofthe invention, being de novo DNA cytosine methyltransferases, areuniquely suited to the latter function, owing to the fact that, unlikemaintenance methyltransferases, their preferred substrate is nothemimethylated DNA.

As exemplified in Examples 7 and 8, isolated polypeptides of theinvention function as in vitro DNA methyltransferases when combined inan appropriately buffered solution with the appropriate cofactors and asubstrate DNA. The substrate DNA may be selected from any naturalsource, e.g., genomic DNA, or a recombinant source such as a DNAfragment amplified by the polymerase chain reaction. The substrate DNAmay be prokaryotic or eukaryotic DNA. In a preferred embodiment, thesubstrate DNA is mammalian DNA, and most preferredly, the substrate DNAis human DNA.

It will be well appreciated by those in the art that in vitromethylation of DNA may be used to direct or regulate the expression ofsaid DNA in a biological system. For example, over-expression,under-expression or lack of expression of a particular native DNAsequence in a host cell or organism may be attributed to the fact thatthe DNA is under-methylated (hypomethylated) or not methylated. Thus, invitro methylation of a recombinant form of said DNA, and the subsequentintroduction of the methylated, recombinant DNA into the cell ororganism, may effect an increase or decrease in the expression of theencoded polypeptide.

Also, it will be readily apparent to the skilled artisan that the invitro methylation pattern will be maintained after introduction into abiological system by the action of maintenance methyltransferasepolypeptides in said system.

In one embodiment of the invention, the biological system selected forthe introduction of in vitro methylated DNA may be prokaryotic oreukaryotic. In a preferred embodiment, the biological system ismammalian, and the most preferred embodiment is when the biologicalsystem is human.

Methods for introducing the in vitro methylated DNA into the biologicalsystem are well known in the art, and the skilled artisan will recognizethat the in vitro methylation of DNA may be a preliminary step to anysystem of gene therapy detailed herein.

VI. Genetic Screening and Diagnostic Assays

To map the human chromosome locations, the GenBank STS database wassearched using Dnmt3a and Dnmt3b sequences as queries. The searchidentified markers WI-6283 (GenBank Accession number G06200) andSHGC-15969 (GenBank Accession number G15302) as matching the cDNAsequence of Dnmt3a and Dnmt3b, respectively. WI-6283 has been mapped to2p23 between D2S171 and D2S174 (48-50 cM) on the radiation hybrid map byWhitehead Institute/MIT Center for Genome Research. The correspondingmouse chromosome location is at 4.0 cM on chromosome 12. SHGC-15969 hasbeen mapped to 20 pl 1.2 between D20S184 and D20S106 (48-50 cM) byStanford Human Genome Center. The corresponding mouse chromosome locusis at 84.0 cM on chromosome 2.

These data are valuable as markers to be correlated with genetic mapdata. Such data are found, for example, in V. McKusick, MendelianInheritance in Man (available on-line through Johns Hopkins, UniversityWelch Medical Library). The relationship between genes and diseases thathave been mapped to the same chromosomal region are then identifiedthrough linkage analysis (coinheritence of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

This invention also relates to the use of de novo DNA cytosinemethyltransferase polynucleotides for use as diagnostic reagents.Detection of a mutated form of a de novo DNA cytosine methyltransferasegene associated with a dysfunction will provide a diagnostic tool thatcan add to or define a diagnosis of a disease or susceptibility to adisease which results from under-expression, over-expression or alteredexpression of the mutated de novo DNA cytosine methyltransferase.Individuals carrying mutations in one or more de novo DNA cytosinemethyltransferase genes may be detected at the DNA level by a variety oftechniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled de novo DNA cytosinemethyltransferase nucleotide sequences. Perfectly matched sequences canbe distinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence differences may alsobe detected by alterations in electrophoretic mobility of DNA fragmentsin gels, with or without denaturing agents, or by direct DNA sequencing(see, e.g., Myers, et al., Science 230:1242 (1985)). Sequence changes atspecific locations may also be revealed by nuclease protection assays,such as RNase and S1 protection or the chemical cleavage method (seeCotton, et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1985)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to neoplastic disorders through detection of mutations inone or more de novo DNA cytosine methyltransferase genes by the methodsdescribed.

In addition, neoplastic disorders may be diagnosed by methods thatdetermine an abnormally decreased or increased level of de novo DNAcytosine methyltransferase polypeptide or de novo DNA cytosinemethyltransferase mRNA in a sample derived from a subject. Decreased orincreased expression may be measured at the RNA level using any of themethods well known in the art for the quantitation of polynucleotides;for example, RT-PCR, RNase protection, Northern blotting and otherhybridization methods may be utilized. Assay techniques that may be usedto determine the level of a protein, such as an de novo DNA cytosinemethyltransferase protein, in a sample derived from a host are wellknown to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western blot analysis andELISA assays.

Additionally, methods are provided for diagnosing or determining asusceptibility of an individual to neoplastic disorders, comprising (a)assaying the de novo DNA cytosine methyltransferase protein geneexpression level in mammalian cells or body fluid; and (b) comparingsaid de novo DNA cytosine methyltransferase protein gene expressionlevel with a standard de novo DNA cytosine methyltransferase proteingene expression level whereby an increase or decrease in said de novoDNA cytosine methyltransferase gene expression level over said standardis indicative of an increased or decreased susceptibility to aneoplastic disorder.

VII. De Novo DNA Cytosine Methyltransferase Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them may also be used as immunogens to produceantibodies immunospecific for the de novo DNA cytosine methyltransferasepolypeptides. By “immunospecific” is meant that the antibodies haveaffinities for the polypeptides of the invention that are substantiallygreater in their affinities for related polypeptides such as theanalogous proteins of the prior art.

Antibodies generated against the de novo DNA cytosine methyltransferasepolypeptides can be obtained by administering the polypeptides orepitope-bearing fragments, analogs or cells to an animal, preferably anonhuman, using routine protocols. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique (Kohler, G. and Milstein, C., Nature 256:495-497(1975)), the trioma technique, the human B-cell hybridoma technique(Kozbor, et al. Immunology Today 4:72 (1983)) and the EBV-hybridomatechnique (Cole, et al., Monoclonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss, Inc., (1985)).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) may also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against de novo DNA cytosine methyltransferase polypeptidesmay also be employed to treat neoplastic disorders, among others.

VIII. Agonist and Antagonist Screening

The de novo DNA cytosine methyltransferase polypeptides of the presentinvention may be employed in a screening process for compounds whichbind one of the proteins and which activate (agonists) or inhibitactivation of (antagonists) one of the polypeptides of the presentinvention. Thus, polypeptides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics (see Coligan, etal., Current Protocols in Immunology 1(2):Chapter 5 (1991)).

By “agonist” is intended naturally occurring and synthetic compoundscapable of enhancing a de novo DNA cytosine methyltransferase activity(e.g., increasing the rate of DNA methylation). By “antagonist” isintended naturally occurring and synthetic compounds capable ofinhibiting a de novo DNA cytosine methyltransferase activity.

DNA methylation is an important, fundamental regulatory mechanism forgene expression, and, therefore, the methylated state of a particularDNA sequence may be associated with many pathologies. Accordingly, it isdesirous to find both compounds and drugs which stimulate de novo DNAcytosine methyltransferase activity and which can inhibit the functionof de novo DNA cytosine methyltransferase protein. In general, agonistsare employed for therapeutic and prophylactic purposes including thetreatment of ceratin types of neoplastic disorders. For example, de novomethylation of growth regulatory genes in somatic tissues is associatedwith tumorigenesis in humans (Laird, P. W. and Jaenisch, R. Ann. Rev.Genet. 30:441-464 (1996); Baylin, S. B. et al., Adv. Cancer. Res.72:141-196 (1998); and Jones, P. A. and Gonzalgo, M. L. Proc. Natl.Acad. Sci. USA 94:2103-2105 (1997)).

In general, such screening procedures involve producing appropriatecells which express the polypeptide of the present invention. Such cellsinclude cells from mammals, yeast, Drosophila or E. coli. Cellsexpressing the protein (or cell membrane containing the expressedprotein) are then contacted with a test compound to observe binding,stimulation or inhibition of a functional response.

Alternatively, the screening procedure may be an in vitro procedure inwhich the activity of isolated DNMT3 protein is tested in the presenceof a potential agonist or antagonist of DNMT3 de novo DNA cytosinemethyltransferase activity. Such in vitro assays are known to thoseskilled in the art, and by way of example are demonstrated in Example 4.

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the protein is detected by means of alabel directly or indirectly associated with the candidate compound orin an assay involving competition with a labeled competitor. Further,these assays may test whether the candidate compound affects activity ofthe protein, using detection systems appropriate to the cells bearingthe protein at their surfaces. Inhibitors of activation are generallyassayed in the presence of a known agonist and the effect on activationby the agonist in the presence of the candidate compound is observed.Standard methods for conducting such screening assays are wellunderstood in the art.

Examples of potential de novo DNA cytosine methyltransferase proteinantagonists include antibodies or, in some cases, oligonucleotides orproteins which are closely related to the substrate of the de novo DNAcytosine methyltransferase protein, e.g., small molecules which bind tothe protein so that the activity of the protein is prevented.

IX. Gene Therapy Applications

For overview of gene therapy, see Strachan, T. & Read A. P., Chapter 20,“Gene Therapy and Other Molecular Genetic-based Therapeutic Approaches,”(and references cited therein) in Human Molecular Genetics, BIOSScientific Publishers Ltd. (1996).

Initial research in the area of gene therapy focused on a fewwell-characterized and highly publicized disorders: cystic fibrosis(Drumm, M. L. et al, Cell 62:1227-1233 (1990); Gregory, R. J. et al.Nature 347:358-363 (1990); Rich, D. P. et al. Nature 347:358-363(1990)); and Gaucher disease (Sorge, J. et al., Proc. Natl. Acad. Sci.(USA) 84:906-909 (1987); Fink, J. K. et al., Proc. Natl. Acad. Sci.(USA) 87:2334-2338 (1990)); and certain forms of hemophilia-Bontempo, F.A. et al., Blood 69:1721-1724 (1987); Palmer, T. D. et al., Blood73:438-445 (1989); Axelrod, J. H. et al. Proc. Natl. Acad. Sci. (USA)87:5173-5177 (1990); Armentano. D. et al., Proc. Natl. Acad. Sci. (USA)87:6141-6145 (1990)); and muscular dystrophy (Partridge, T. A. et al.,Nature 337:176-179 (1989); Law, P. K. et al., Lancet 336:114-115 (1990);Morgan, J. E. et al., J. Cell Biol. 111:2437-2449 (1990)).

More recently, the application of gene therapy in the treatment of awider variety of disorders is progressing, for example: cancer(Runnebaum, I. B., Anticancer Res. 17(4B): 2887-2890, (1997)), heartdisease (Rader, D. J., Int. J. Clin. Lab. Res. 27(1): 35-43, (1997);Malosky, S., Curr. Opin. Cardiol. 11(4): 361-368, (1996)), centralnervous system disorders and injuries (Yang, K., et al., Neurotrauma J.14(5): 281-297, (1997); Zlokovic. B. V. et al. Neurosurgery 40(4):789-803, (1997); Zlokovic, B. V., et al. Neurosurgery 40(4): 805-812,(1997)), vascular diseases (Clowes, A. W., Thromb. Haemost. 78(1):605-610, 1997), muscle disorders (Douglas. J. T., et al., Neuromuscul.Disord. 7(5): 284-298, (1997); Huard, J., et al., Neuromuscul. Disord7(5): 299-313, (1997)), rheumatoid arthritis (Evans, C. H., et al.,Curr. Opin. Rheumatol. 8(3): 230-234, (1996)) and epithelial tissuedisorders (Greenhalgh, D. A., et al., Invest Dermatol. J. 103(5 Suppl.):63S-93S, (1994)).

In a preferred approach, one or more isolated nucleic acid molecules ofthe invention are introduced into or administered to the animal. Suchisolated nucleic acid molecules may be incorporated into a vector orvirion suitable for introducing the nucleic acid molecules into thecells or tissues of the animal to be treated, to form a transfectionvector. Techniques for the formation of vectors or virions comprisingthe de novo DNA cytosine methyltransferase-encoding nucleic acidmolecules are well known in the art and are generally described in“Working Toward Human Gene Therapy,” Chapter 28 in Recombinant DNA, 2ndEd., Watson, J. D. et al., eds., New York: Scientific American Books,pp. 567-581 (1992). An overview of suitable vectors or virions isprovided in an article by Wilson, J. M. (Clin. Exp. Immunol. 107(Suppl.1): 31-32, (1997)). Such vectors are derived from viruses that containRNA (Vile, R. G., et al., Br. Med Bull. 51(1): 12-30, (1995)) or DNA(Ali M. et al. Gene Ther. 1(6): 367-384, (1994)). Example vector systemsutilized in the art include the following: retroviruses (Vile, R. G.supra.), adenoviruses (Brody. S. L. et al., Ann. N.Y. Acad. Sci. 716:90-101, (1994)), adenoviral/retroviral chimeras (Bilbao, G., et al.,FASEB J. 11 (8): 624-634, (1997)), adeno-associated viruses (Flotte, T.R. and Carter, B. J., Gene Ther. 2(6): 357-362, (1995)), herpes simplexvirus (Latchman, D. S., Mol. Biotechnol. 2(2): 179-195, (1994)),Parvovirus (Shaughnessy, E., et al., Semin Oncol. 23(1): 159-171.(1996)) and reticuloendotheliosis virus (Donburg, R., Gene Therap. 2(5):301-310, (1995)). Also of interest in the art, the development ofextrachromosomal replicating vectors for gene therapy (Calos, M. P.,Trends Genet. 12(11): 463-466, (1996)).

Other, nonviral methods for gene transfer known in the art (Abdallah, B.et al., Biol. Cell 85(1): 1-7, (1995)) might be utilized for theintroduction of de novo DNA cytosine methyltransferase polynucleotidesinto target cells; for example, receptor-mediated DNA delivery (Philips,S. C., Biologicals 23(1): 13-16, (1995)) and lipidic vector systems(Lee, R. J. and Huang, L., Crit. Rev. Ther. Drug Carrier Syst. 14(2):173-206, (1997)) are promising alternatives to viral-based deliverysystems.

General methods for construction of gene therapy vectors and theintroduction thereof into affected animals for therapeutic purposes maybe obtained in the above-referenced publications, the disclosures ofwhich are specifically incorporated herein by reference in theirentirety. In one such general method, vectors comprising the isolatedpolynucleotides of the present invention are directly introduced intotarget cells or tissues of the affected animal, preferably by injection,inhalation, ingestion or introduction into a mucous membrane viasolution; such an approach is generally referred to as “in vivo” genetherapy. Alternatively, cells, tissues or organs may be removed from theaffected animal and placed into culture according to methods that arewell-known to one of ordinary skill in the art; the vectors comprisingthe de novo DNA cytosine methyltransferase polynucleotides may then beintroduced into these cells or tissues by any of the methods describedgenerally above for introducing isolated polynucleotides into a cell ortissue, and, after a sufficient amount of time to allow incorporation ofthe de novo DNA cytosine methyltransferase polynucleotides, the cells ortissues may then be re-inserted into the affected animal. Since theintroduction of a De novo DNA cytosine methyltransferase gene isperformed outside of the body of the affected animal, this approach isgenerally referred to as “ex vivo” gene therapy.

For both in vivo and ex vivo gene therapy, the isolated de novo DNAcytosine methyltransferase polynucleotides of the invention mayalternatively be operatively linked to a regulatory DNA sequence, whichmay be a de novo DNA cytosine methyltransferase promoter or an enhancer,or a heterologous regulatory DNA sequence such as a promoter or enhancerderived from a different gene, cell or organism, to form a geneticconstruct as described above. This genetic construct may then beinserted into a vector, which is then used in a gene therapy protocol.The need for transcriptionally targeted and regulatable vectorsproviding cell-type specific and inducible promoters is well recognizedin the art (Miller, N. and Whelan, J., Hum. Gene Therap. 8(7): 803-815,(1997); and Walther, W. and Stein, U., Mol. Med. J, 74(7): 379-392,(1996)), and for the purposes of de novo DNA cytosine methyltransferasegene therapy, is incorporated herein by reference.

The construct/vector may be introduced into the animal by an in vivogene therapy approach, e.g., by direct injection into the target tissue,or into the cells or tissues of the affected animal in an ex vivoapproach. In another preferred embodiment, the genetic construct of theinvention may be introduced into the cells or tissues of the animal,either in vivo or ex vivo, in a molecular conjugate with a virus (e.g.,an adenovirus or an adeno-associated virus) or viral components (e.g.,viral capsid proteins; see WO 93/07283). Alternatively, transfected hostcells, which may be homologous or heterologous, may be encapsulatedwithin a semi-permeable barrier device and implanted into the affectedanimal, allowing passage of de novo DNA cytosine methyltransferasepolypeptides into the tissues and circulation of the animal butpreventing contact between the animal's immune system and thetransfected cells (see WO 93/09222). These approaches result inincreased production of de novo DNA cytosine methyltransferase by thetreated animal via (a) random insertion of the de novo DNA cytosinemethyltransferase gene into the host cell genome; or (b) incorporationof the de novo DNA cytosine methyltransferase gene into the nucleus ofthe cells where it may exist as an extrachromosomal genetic element.General descriptions of such methods and approaches to gene therapy maybe found, for example, in U.S. Pat. No. 5,578,461, WO 94/12650 and WO93/09222.

Antisense oligonucleotides have been described as naturally occurringbiological inhibitors of gene expression in both prokaryotes (Mizuno etal., Proc. Natl. Acad. Sci. USA 81:1966-1970 (1984)) and eukaryotes(Heywood, Nucleic Acids Res. 14:6771-6772 (1986)), and these sequencespresumably function by hybridizing to complementary mRNA sequences,resulting in hybridization arrest of translation (Paterson, et al.,Proc. Natl. Acad. Sci. USA, 74:4370-4374 (1987)).

Thus, another gene therapy approach utilizes antisense technology.Antisense oligonucleotides are short synthetic DNA or RNA nucleotidemolecules formulated to be complementary to a specific gene or RNAmessage. Through the binding of these oligomers to a target DNA or mRNAsequence, transcription or translation of the gene can be selectivelyblocked and the disease process generated by that gene can be halted(see, for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press (1989)). The cytoplasmiclocation of mRNA provides a target considered to be readily accessibleto antisense oligodeoxynucleotides entering the cell; hence much of thework in the field has focused on RNA as a target. Currently, the use ofantisense oligodeoxynucleotides provides a useful tool for exploringregulation of gene expression in vitro and in tissue culture(Rothenberg, et al, J. Natl. Cancer Inst. 81:1539-1544 (1989)).

Antisense therapy is the administration of exogenous oligonucleotideswhich bind to a target polynucleotide located within the cells. Forexample, antisense oligonucleotides may be administered systemically foranticancer therapy (Smith, International Application Publication No. WO90/09180).

The antisense oligonucleotides of the present invention includederivatives such as S-oligonucleotides (phosphorothioate derivatives orS-oligos, see, Jack Cohen, supra). S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention may beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide which is a sulfur transferreagent. See Iyer et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer etal., J. Am. Chem. Soc. 112:1253-1254 (1990), the disclosures of whichare fully incorporated by reference herein.

As described herein, sequence analysis of SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3 or the SEQ ID NO:4 cDNA clone shows that sequence that isnonhomologous to known DNA methyltransferase sequences may be identified(see FIGS. 1 and 4). Thus, the antisense oligonucleotides of the presentinvention may be RNA or DNA that is complementary to and stablyhybridize with such sequences that are specific for a de novo DNAcytosine methyltransferase gene of the invention. Use of anoligonucleotide complementary to such regions allows for selectivehybridization to a de novo DNA cytosine methyltransferase mRNA and notto an mRNA encoding a maintenance methyltransferase protein.

Preferably, the antisense oligonucleotides of the present invention area 15 to 30-mer fragment of the antisense DNA molecule coding for uniquesequences of the de novo DNA cytosine methyltransferase cDNAs. Preferredantisense oligonucleotides bind to the 5′-end of the de novo DNAcytosine methyltransferase mRNAs. Such antisense oligonucleotides may beused to down regulate or inhibit expression of the gene.

Other criteria that are known in the art may be used to select theantisense oligonucleotides, varying the length or the annealing positionin the targeted sequence.

Included as well in the present invention are pharmaceuticalcompositions comprising an effective amount of at least one of theantisense oligonucleotides of the invention in combination with apharmaceutically acceptable carrier. In one embodiment, a singleantisense oligonucleotide is utilized.

In another embodiment, two antisense oligonucleotides are utilized whichare complementary to adjacent regions of the genome. Administration oftwo antisense oligonucleotides that are complementary to adjacentregions of the genome or corresponding mRNA may allow for more efficientinhibition of genomic transcription or mRNA translation, resulting inmore effective inhibition of protein or mRNA production.

Preferably, the antisense oligonucleotide is coadministered with anagent which enhances the uptake of the antisense molecule by the cells.For example, the antisense oligonucleotide may be combined with alipophilic cationic compound which may be in the form of liposomes. Theuse of liposomes to introduce nucleotides into cells is taught, forexample, in U.S. Pat. Nos. 4,897,355 and 4,394,448, the disclosures ofwhich are incorporated by reference in their entirety (see also U.S.Pat. Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788, 4,673,567,4,247,411, and 4,814,270 for general methods of preparing liposomescomprising biological materials).

Alternatively, the antisense oligonucleotide may be combined with alipophilic carrier such as any one of a number of sterols includingcholesterol, cholate and deoxycholic acid. A preferred sterol ischolesterol.

In addition, the antisense oligonucleotide may be conjugated to apeptide that is ingested by cells. Examples of useful peptides includepeptide hormones, antigens or antibodies, and peptide toxins. Bychoosing a peptide that is selectively taken up by the targeted tissueor cells, specific delivery of the antisense agent may be effected. Theantisense oligonucleotide may be covalently bound via the 5′OH group byformation of an activated aminoalkyl derivative. The peptide of choicemay then be covalently attached to the activated antisenseoligonucleotide via an amino and sulfhydryl reactive hetero bifunctionalreagent. The latter is bound to a cysteine residue present in thepeptide. Upon exposure of cells to the antisense oligonucleotide boundto the peptide, the peptidyl antisense agent is endocytosed and theantisense oligonucleotide binds to the target mRNA to inhibittranslation (Haralambid et al., WO 8903849 and Lebleu et al., EP0263740).

The antisense oligonucleotides and the pharmaceutical compositions ofthe present invention may be administered by any means that achievetheir intended purpose. For example, administration may be byparenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,or transdermal routes. The dosage administered will be dependent uponthe age, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired.

Compositions within the scope of this invention include all compositionswherein the antisense oligonucleotide is contained in an amounteffective to achieve the desired effect, for example, inhibition ofproliferation and/or stimulation of differentiation of the subjectcancer cells. While individual needs vary, determination of optimalranges of effective amounts of each component is with the skill of theart.

Alternatively, antisense oligonucleotides can be prepared which aredesigned to interfere with transcription of the gene by bindingtranscribed regions of duplex DNA (including introns, exons, or both)and forming triple helices (e.g., see Froehler et al. WO 91/06626 orToole, WO 92/10590). Preferred oligonucleotides for triple helixformation are oligonucleotides which have inverted polarities for atleast two regions of the oligonucleotide (Id.). Such oligonucleotidescomprise tandem sequences of opposite polarity such as3′---5′-L-5′---3′, or 5′---3′-L-3′---5′, wherein L represents a 0-10base oligonucleotide linkage between oligonucleotides. The invertedpolarity form stabilizes single-stranded oligonucleotides to exonucleasedegradation (Froehler et al., supra). The criteria for selecting suchinverted polarity oligonucleotides is known in the art, and suchpreferred triple helix-forming oligonucleotides of the invention arebased upon SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In therapeutic application, the triple helix-forming oligonucleotidescan be formulated in pharmaceutical preparations for a variety of modesof administration, including systemic or localized administration, asdescribed above.

The antisense oligonucleotides of the present invention may be preparedaccording to any of the methods that are well known to those of ordinaryskill in the art, as described above.

Ribozymes provide an alternative method to inhibit mRNA function.Ribozymes may be RNA enzymes, self-splicing RNAs, and self-cleaving RNAs(Cech et al., Journal of Biological Chemistry 267:17479-17482 (1992)).It is possible to construct de novo ribozymes which have an endonucleaseactivity directed in trans to a certain target sequence. Since theseribozymes can act on various sequences, ribozymes can be designed forvirtually any RNA substrate. Thus, ribozymes are very flexible tools forinhibiting the expression of specific genes and provide an alternativeto antisense constructs.

A ribozyme against chloramphenicol acetyltransferase mRNA has beensuccessfully constructed (Haseloffet al., Nature 334:585-591 (1988);Uhlenbeck et al., Nature 328:596-600 (1987)). The ribozyme containsthree structural domains: 1) a highly conserved region of nucleotideswhich flank the cleavage site in the 5′ direction; 2) the highlyconserved sequences contained in naturally occurring cleavage domains ofribozymes, forming a base-paired stem; and 3) the regions which flankthe cleavage site on both sides and ensure the exact arrangement of theribozyme in relation to the cleavage site and the cohesion of thesubstrate and enzyme. RNA enzymes constructed according to this modelhave already proved suitable in vitro for the specific cleaving of RNAsequences (Haseloff et al., supra).

Alternatively, hairpin ribozymes may be used in which the active site isderived from the minus strand of the satellite RNA of tobacco ring spotvirus (Hampel et al., Biochemistry 28:4929-4933 (1989)). Recently, ahairpin ribozyme was designed which cleaves human immunodeficiency virustype I RNA (Ojwang et al. Proc. Natl. Acad. Sci. USA 89:10802-10806(1992)). Other self-cleaving RNA activities are associated withhepatitis delta virus (Kuo et al., J. Virol. 62:4429-4444 (1988)).

As discussed above, preferred targets for ribozymes are the de novo DNAcytosine methyltransferase nucleotide sequences that are not homologouswith maintenance methyltransferase sequences such as Dnmt 1 or Dnmt 2.Preferably, the ribozyme molecule of the present invention is designedbased upon the chloramphenicol acetyltransferase ribozyme or hairpinribozymes, described above. Alternatively, ribozyme molecules aredesigned as described by Eckstein et al. (International Publication No.WO 92/07065) who disclose catalytically active ribozyme constructionswhich have increased stability against chemical and enzymaticdegradation, and thus are useful as therapeutic agents.

In an alternative approach, an external guide sequence (EGS) can beconstructed for directing the endogenous ribozyme, RNase P, tointracellular mRNA, which is subsequently cleaved by the cellularribozyme (Altman et al., U.S. Pat. No. 5,168,053). Preferably, the EGScomprises a ten to fifteen nucleotide sequence complementary to an mRNAand a 3′-NCCA nucleotide sequence, wherein N is preferably a purine(Id.). After EGS molecules are delivered to cells, as described below,the molecules bind to the targeted mRNA species by forming base pairsbetween the mRNA and the complementary EGS sequences, thus promotingcleavage of mRNA by RNase P at the nucleotide at the 5′side of thebase-paired region (Id.).

Included as well in the present invention are pharmaceuticalcompositions comprising an effective amount of at least one ribozyme orEGS of the invention in combination with a pharmaceutically acceptablecarrier. Preferably, the ribozyme or EGS is coadministered with an agentwhich enhances the uptake of the ribozyme or EGS molecule by the cells.For example, the ribozyme or EGS may be combined with a lipophiliccationic compound which may be in the form of liposomes, as describedabove. Alternatively, the ribozyme or EGS may be combined with alipophilic carrier such as any one of a number of sterols includingcholesterol, cholate and deoxycholic acid. A preferred sterol ischolesterol.

The ribozyme or EGS, and the pharmaceutical compositions of the presentinvention may be administered by any means that achieve their intendedpurpose. For example, administration may be by parenteral, subcutaneous,intravenous, intramuscular, intra-peritoneal, or transdermal routes. Thedosage administered will be dependent upon the age, health, and weightof the recipient, kind of concurrent treatment, if any, frequency oftreatment, and the nature of the effect desired. For example, as much as700 milligrams of antisense oligodeoxynucleotide has been administeredintravenously to a patient over a course of 10 days (i.e., 0.05mg/kg/hour) without signs of toxicity (Sterling, “Systemic AntisenseTreatment Reported,” Genetic Engineering News 12(12):1, 28 (1992)).

Compositions within the scope of this invention include all compositionswherein the ribozyme or EGS is contained in an amount which is effectiveto achieve inhibition of proliferation and/or stimulate differentiationof the subject cancer cells, or alleviate AD. While individual needsvary, determination of optimal ranges of effective amounts of eachcomponent is with the skill of the art.

In addition to administering the antisense oligonucleotides, ribozymes,or EGS as a raw chemical in solution, the therapeutic molecules may beadministered as part of a pharmaceutical preparation containing suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the antisenseoligonucleotide, ribozyme, or EGS into preparations which can be usedpharmaceutically.

Suitable formulations for parenteral administration include aqueoussolutions of the antisense oligonucleotides, ribozymes, EGS inwater-soluble form, for example, water-soluble salts. In addition,suspensions of the active compounds as appropriate oily injectionsuspensions may be administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, ethyl oleate or triglycerides. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

Alternatively, antisense RNA molecules, ribozymes, and EGS can be codedby DNA constructs which are administered in the form of virions, whichare preferably incapable of replicating in vivo (see, for example,Taylor, WO 92/06693). For example, such DNA constructs may beadministered using herpes-based viruses (Gage et al., U.S. Pat. No.5,082,670). Alternatively, antisense RNA sequences, ribozymes, and EGScan be coded by RNA constructs which are administered in the form ofvirions, such as retroviruses. The preparation of retroviral vectors iswell known in the art (see, for example, Brown et al., “RetroviralVectors,” in DNA Cloning: A Practical Approach, Volume 3, IRL Press,Washington, D.C. (1987)).

Specificity for gene expression may be conferred by using appropriatecell-specific regulatory sequences, such as cell-specific enhancers andpromoters. Such regulatory elements are known in the art, and their useenables therapies designed to target specific tissues, such as liver,lung, prostate, kidney, pancreas, etc., or cell populations, such aslymphocytes, neurons, mesenchymal, epithelial, muscle, etc.

In addition to the above noted methods for inhibiting the expression ofthe de novo methyltransferase genes of the invention, gene therapeuticapplications may be employed to provide expression of the polypeptidesof the invention.

EXAMPLES Example 1 Cloning and Sequence Analysis of the Mouse Dnmt3a andDnmt3b and the Human DNMT3A and DNMT3B Genes and Polypeptides

In search of a mammalian de novo DNA methyltransferase, two independentapproaches were undertaken, based on the assumption that an unknownmammalian DNA methyltransferase must contain the highly conservedcytosine methyltransferase motifs in the catalytic domain of knownmethyltransferases (Lauster, R. et al., J. Mol. Biol. 206:305-312 (1989)and Kumar. S. et al. Nucl. Acids Res. 22:1-10 (1994)). Our firstapproach, an RT/PCR-based screening using oligonucleotide primerscorresponding to the conserved motifs of the known cytosine DNAmethyltransferases, failed to detect any novel methyltransferase genefrom Dnmt1 null ES cells (data not shown).

The second approach was a tblastn search of the dbEST database usingfull length bacterial cytosine methyltransferase sequences as queries.

A search of the dbEST database was performed with the tblastn program(Altschul, S. F. et al, J. Mol. Biol. 215:403-410 (1990)) usingbacterial cytosine methyltransferases as queries. Candidate ESTsequences were used one by one as queries to search the non-redundantprotein sequence database in GenBank with the blastx program. Thisprocess would eliminate EST clones corresponding to known genes(including known DNA methyltransferases) and those which show a highersimilarity to other sequences than to DNA methyltransferases. Two ESTclones (GenBank numbers W76111 and N88352) were found after the initialsearch. Two more EST sequences (f12227 and T66356) were later foundafter a blastn search of dbEST with the EST sequence of W76111 as aquery. Two of the EST clones (W76111 and T66356) were deposited by theI.M.A.G.E. Consortium (Lawrence Livermore National Laboratory,Livermore, Calif.) and obtained from American Type Culture Collection(Manassas, Va.). Sequencing of these two cDNA clones revealed that theywere partial cDNA clones with large open reading frames corresponding totwo related genes. The translated amino acid sequences revealed thepresence of the highly conserved motifs characteristic of DNA cytosinemethyltransferases. The EST sequences were then used as probes forscreening mouse E7.5 embryo and ES cell cDNA libraries and a human heartcDNA library (Clontech, Calif.).

In a screening of the dbEST database using 35 bacterial cytosine-5 DNAmethyltransferase sequences as queries, eight EST clones were found tohave the highest similarity but not to be identical to the knowncytosine-5-DNA methyltransferase genes. Six of the eight EST sequenceswere deposited by the I.M.A.G.E. Consortium (Lawrence Livermore NationalLaboratory, Livermore, Calif.) and obtained from TIGR/ATCC (AmericanType Culture Collection, Manassas, Va.). Sequencing of these 6 cDNAclones revealed that they were partial cDNA clones with large openreading frames corresponding to three novel genes. The translated aminoacid sequences revealed the presence of the highly conserved motifscharacteristic of DNA cytosine methyltransferases. The EST sequenceswere then used as probes for screening a mouse ES cell cDNA library, amouse E11.5 embryonic cDNA library (Clontech, Calif.) and human heartcDNA library.

Human and mouse cDNA libraries were screened using EST sequences asprobes. Sequencing analysis of several independent cDNA clones revealedthat two homologous genes were present in both human and mouse. This wasfurther confirmed by Southern analysis of genomic DNA, intron/exonmapping and sequencing of genomic DNA (data not shown). The full lengthmouse cDNAs for each gene were assembled and complete sequencingrevealed that both genes contained the highly conserved cytosinemethyltransferase motifs and shared overall 51% of amino acid identity(76% identity in the catalytic domain) (FIG. 3). Since these two genesshowed little sequence similarities to Dnmt1(Bestor, T. H. et al, J.Mol. Biol. 203:971-983 (1988) and Yen, R-W. C. et al., Nucleic AcidsRes. 20:2287-2291 (1992)) and a recently cloned putative DNAmethyltransferase gene, Dnmt2 (see Yoder, J. A. and Bestor, T. H. Hum.Mol. Genet. 7:279-284 (1998)) and Okano, M., Xie, S. and Li, E.,(submitted)), beyond the conserved methyltransferase motifs in thecatalytic domain, they were named Dnmt3a and Dnmt3b.

The full length Dnmt3a and Dnmt3b genes encode 908 and 859 amino acidpolypeptides, termed Dnmt3a and Dnmt3b1, respectively. Nucleotide andamino acid sequences of each are presented in FIGS. 1A, 1B, 2A, and 2B.The Dnmt3b gene also produces through alternative splicing at least twoshorter isoforms of 840 and 777 amino acid residues, termed Dnmt3b2 andDnmt3b3, respectively, (FIG. 4).

To obtain full length human cDNA, fetal heart and fetal testis cDNAlibraries were screened using EST clones as probes. Sequencing analysisof several overlapping DNMT3A cDNA clones indicates that the DNMT3A geneencodes a polypeptide of 912 amino acid residues. DNMT3 B cDNA cloneswere not detected in the fetal heart library, but several DNMT3B cDNAclones were obtained after screening the fetal testis library. PCRscreening of large cDNA clones from 24 human tissues was also performedusing the Human Rapid-Screen™ cDNA Library Panels (OriGene Technologies,MD). The largest cDNA clone contained a 4.2 kb insert from a smallintestine cDNA library. Sequencing analysis of overlapping cDNA clonesindicated that the deduced full length DMNT3B consists of 853 amino acidresidues. Since in-frame stop codons are found upstream of the ATG ofboth DNMT3A and DNMT3B, it is concluded that these cDNA clones encodefull-length DNMT3A and DNMT3B proteins.

The full length human DNMT3A and DNMT3B cDNAs encode 912 and 853 aminoacid polypeptides, termed DNMT3A and DNMT3B1, respectively. Nucleotideand polypeptide sequences are presented in FIGS. 1C, 1D, 2C and 2D,respectively. The DNMT3B gene also produces through alternative splicingat least two shorter isoforms, termed DNMT3B2 and DNMT3B3, respectively.DNMT3B2 comprises amino acid residues 1 to 355 and 376 to 853 of SEQ IDNO:4; and DNMT3B 3 comprises amino acid residues 1 to 355 and 376 to 743and 807 to 853 of SEQ ID NO:4.

Also identified through screening was a related zebrafish gene, termedZmt-3, which from the EST database (GenBank number AF135438).

The GenBank STS database was used to map chromosome localization byusing DNMT3A and DNMT3B sequences as queries. The results identifiedmarkers WI-6283 (GenBank Accession number G06200) and SHGC-15969(GenBank Accession number G15302), which matched the cDNA sequence ofDNMT3A and DNMT3B, respectively. WI-6283 has been mapped to 2p23 betweenD2S171 and D2S174 (48-50 cM) on the radiation hybrid map by WhiteheadInstitute/MIT Center for Genome Research. The corresponding mousechromosome location is at 4.0 cM on chromosome 12. SHGC-15969 has beenmapped to 20 pl 1.2 between D20S184 and D20S106 (48-50 cM) by StanfordHuman Genome Center. The corresponding mouse chromosome locus is at 84.0cM on chromosome 2.

Taking the advantage of the newly identified DNMT3A and DNMT3B cDNAsequences, the human genomic sequence database was searched by BLAST.While human DNMT3A cDNA did not match any related genomic sequences inthe database, a DNMT3B genomic YAC clone from GenBank (AL035071) wasidentified when DNMT3B cDNA sequences were used as queries.

The DNMT3B cDNA and the genomic DNA GenBank (AL035071) clone were usedto map all exons using BESTFIT of the GCG program. As shown in FIG. 4C,there are total 23 exons, spanning some 48 kb genomic DNA. The putativefirst exon is located within a CpG island where the promoter is probablylocated as predicted by the GENSCAN program (Whitehead/MIT Center forGenome Research).

Sequencing of various cDNA clones indicates that the human DNMT3B genecontains three alternatively spliced exons, exons 10, 21 and 22. Similarto the mouse gene, DNMT3B1 contains all 23 exons, whereas DNMT3B2 lacksexon 10 and DNMT3B3 lacks exons 10, 21 and 22. The nucleotide sequencesat the exon/intron boundaries are shown in FIG. 4D. The elucidation ofhuman DNMT3B gene structure may facilitate analysis of DNMT3B mutationsin certain cancers with characteristic hypomethylation of genomic: DNA(Narayan, A., et al., Int. J. Cancer 77:833-838 (1998); Qu, G., et al.,Mutan. Res. 423:91-101 (1999)).

FIG. 3A presents an alignment of mouse Dnmt3a and Dnmt3b polypeptidesequences that was accomplished using the GCG program. The verticallines indicate amino acid identity, while the dots and the colonsindicate similarities. Dots in amino acid sequences indicate gapsintroduced to maximize alignment. The conserved Cys-rich region isshaded. The full length mouse Dnmt3a and Dnmt3b genes encode 908 and 859amino acid polypeptides. Furthermore, the analysis reveals that bothgenes contained the highly conserved cytosine methyltransferase motifsand share overall 51% of amino acid identity (76% identity in thecatalytic domain). The Dnmt3b gene also produces at least two shorterisoforms of 840 and 777 amino acid residues, termed Dnmt3b2 and Dnmt3b3,respectively, through alternative splicing (FIG. 4).

FIG. 3B presents a GCG program alignment using the of the proteinsequences of human DNMT3A and DNMT3B1. Vertical lines representidentical amino acid residues, whereas dots represent conserved changes.Dots in amino acid sequences indicate gaps introduced to maximizealignment.

In FIG. 4A, presents a schematic diagram of the overall proteinstructures for mouse Dnmt1, mouse Dnmt2, a putative methyltransferase,and the family of Dnmt3a and Dnmt3b(1-3) methyltransferases. Dnmt1,Dnmt3a and Dnmt3bs all have a putative N-terminal regulatory domain. Thefilled bars represent the five conserved methyltransferase motifs (1,IV, VI, IX, and X). The shaded boxes in Dnmt3a and Dnmt3bs represent theCys-rich region that shows no sequence homology to the Cys-rich,Zn²⁺-binding region of Dnmt1 polypeptide. Sites of alternative splicingat amino acid residues 362-383 and 749-813 in Dnmt3bs are indicated.

An analysis of the human DNMT3 proteins provides similar results as withthe mouse Dnmt proteins. FIG. 4B presents a similar schematic of thehuman DNMT3 proteins and zebrafish Znmt3 protein. The homology betweendifferences between these DNMT3 proteins is indicated by the percentageof sequence identity when compared to DNMT3A.

In addition, the genomic organization of the human DNMT3B1 locus ispresented in FIG. 4C as possessing 23 exons (filled rectangles), a CpGisland (dotted rectangle), a translation initiation codon (ATG) and astop codon (TAG) in exons 2 and 23, respectively. FIG. 4D presents thesize of the exons and introns as well as sequences (uppercase for exonsand lowercase for introns) at exon/intron boundaries.

In FIG. 5, sequence analysis of the catalytic domain indicates that thisnew family of DNA methyltransferases contains conserved amino acidresidues in each of the five highly conserved motifs, but significantdifferences are discernible when compared to the known consensussequences.

FIG. 5A presents an alignment by ClustalW 1.7 of the amino acidsequences of the five highly conserved motifs in eukaryoticmethyltransferase genes. Amino acid residues which are conserved in fiveor more genes are highlighted. The Dnmt3 family methyltransferases aremost closely related to a bacterial DNA methyltransferase (M. Spr.).Sequence comparison of the catalytic domain of all known eukaryotic DNAmethyltransferases and most of the bacterial cytosine methyltransferasesused in the tblastn search indicates that this family ofmethyltransferases are distantly related to all the known eukaryotic DNAmethyltransferases, including the Dnmt 1 polypeptide from vertebrate andplant (Bestor, T. H. et al, J. Mol. Biol. 203:971-983 (1988), Yen, R-W.C. et al., Nucleic Acids Res. 20:2287-2291 (1992) and Finnegan, E. J.and Dennis, E. S. Nucleic Acids Res. 21:2383-2388 (1993)); the human andmouse Dnmt 2 polypeptides (Yoder. J. A. and Bestor, T. H. Hum. Mol.Genet. 7:279-284 (1998), Okano, M., Xie, S. & Li, E., (submitted)); andmasc1 from Ascobolus (Malagnac, F. et al., Cell 91:281-290 (1997)),indicating that the Dnmt3 gene family originated from a uniqueprokaryotic prototype DNA methyltransferase during evolution.

The cysteine-rich region located upstream of the catalytic domain wasfound to be conserved among all of the DNMT3 proteins (FIG. 5B). ThisCysteine-rich region, however, is unrelated to the Cysteine-rich (orZn²⁺-binding) region of DNMT1 (Bestor, T. H. et al., J. Mo. Biol.203:971-983 (1998); Bestor, T. H., EMBO J. 11:2611-2617 (1992)).Interestingly, the Cysteine-rich domain of DNMT3 proteins shareshomology with a similar domain found in the X-linked ATRX gene of theSNF2/SWI family (Picketts, D. J., et al., Hum. Mol. Genet. 5:1899-1907(1996)), raising the interesting possibility that this domain maymediate protein-protein or protein-DNA interactions.

The evolutionary relatedness of cytosine-5 methyltransferases as shownby a non-rooted phylogenic tree is presented in FIG. 5C. Amino acidsequences from motifI to motifVI of bacterial and eukaryotic cytosine-5methyltransferases were used for sequence alignment, and the alignmentdata was analyzed by ClustalW 1.7 under conditions excluding positionswith gaps. Results were visualized utilizing Phlip version 3.3. Aminoacid sequences from motif 1× to motif X were also analyzed and providedsimilar results (data not shown). (Abbreviation Ath; Arabidopsisthaliana, Urc; sea urchin. Xen; Xenopus laevis).

Example 2 Baculovirus-Mediated Expression of Dnmt3a and Dnmt3b

To test whether the newly cloned Dnmt3 genes encode active DNAmethyltransferases, the cDNAs of Dnmt3a, Dnmt3b1, Dnmt3b2, and Dnmt1were overexpressed in insect cells using the baculovirus-mediatedexpression system (Clontech, Calif.).

To construct the Dnmt3a expression vector, pSX134, the Xma I/Eco RIfragment of Dnmt3a cDNA was first cloned into the Nco I/Eco RI sites ofpET2 Id with the addition of an Xma I/Nco I adapter (SX165:5′-CATGGGCAGCAGCCATCATCATCATCATCATGGGAATTCCATGCCC TCCAGCGGCC and SX166:5′-CCGGGGCCGCTGGAGGGCATGGA ATTCCCATGATGATGATGATGATGGCTGCTGCC) thatproduced pSX132His. pSX134 was obtained by cloning the EcoR I/Xba Ifragment of pSX 132His into the EcoR I/Xba I sites of pBacPAK9. TheDnmt3b1 and Dnmt3b2 expression vectors, pSX153 and pSX154, wereconstructed by cloning Eco RI fragments of Dnmt3b1 and Dnmt3b2 cDNA intothe Eco RI site of pBacPAK9, respectively. The Dnmt1 expression vectorpSX148 was constructed by cloning the Bgl I/Sac I fragment of Dnmt1 cDNAinto the Bgl II/Sac I sites of pBacPAK-His2 with the addition of a BglI/Bgl II adapter (SX180:5′-GATCTATGCCAGCGCGAACAGCTCCAGCCCGAGTGCCTGCGCTTGC CTCCC and SX181:5′-AGGCAAGCGCAGGCACTCGGGCTGGAGCTGTT CGCGCTGGCATA).

pSX134 (Dnmt3a), pSX153 (Dnmt3b1), pSX153 (Dnmt3b2) and pSX148 (Dnmt1)were used to make the recombinant baculoviruses according to theprocedures recommended by the manufacturer. T175 flasks were used forcell culture and virus infection. Sf621 host cells were grown in theSF-900 II SFM medium with 10% of the certified FBS (both from GIBCO, MD)and infected with the recombinant viruses 12-24 hours after the cellswere split when they reached 90-95% affluence. After 3 days, theinfected insect cells were harvested and frozen in the liquid nitrogenfor future use.

Example 3 RNA Expression Analysis

ES cells were routinely cultured on a feeder layer of mouse embryonicfibroblasts in DMEM medium containing LIF (500 units/ml) and weredifferentiated as embryoid bodies in suspension culture as described(Lei, H., et al., Development 122:3195-3205 (1996)). Ten days afterseeding, embryoid bodies were harvested for RNA preparation.

Total RNA was prepared from ES cells, ovary and testis tissue using theGTC-CsCl centrifugation method, fractionated on a formaldehydedenaturing 1% agarose gel by electrophoresis and transferred to a nylonmembrane. PolyA+ RNA blots (2 μg per lane) of mouse and human tissueswere obtained from Clontech, Calif. All blots were hybridized torandom-primed cDNA probes in hybridization solution containing 50%formamide at 42° C. and washed with 0.2×SSC, 0.1% SDS at 65° C. andexposed to X-ray film (Kodak).

FIG. 6A presents mouse polyA+ RNA blots of adult tissues (left) andembryos (right) probed with full length Dnmt3a, Dnmt3b and a controlβ-actin cDNA probe. Each lane contains 2 μg of polyA+RNA. (Ht. Heart;Br. Brain; Sp, Spleen; Lu, Lung; Li, Liver; Mu, Skeletal Muscle; Ki.Kidney; Te, Testis; and embryos at gestation days 7 (E7), 11 (E11), 15(E15), and 17 (E17). FIG. 6B is a mouse total RNA blot (10 μg per lane)of ES cell and adult organ RNA samples and FIG. 6C shows a mouse totalRNA blot (20 μg per lane) of undifferentiated (Undiff.) anddifferentiated (Diff.) ES cells RNA hybridized to Dnmt3a, Dnmt3b orβ-actin probes.

It has been shown that the maintenance methylation activity isconstitutively present in proliferating cells, whereas the de novomethylation activity is highly regulated. Active de novo methylation hasbeen shown to occur primarily in ES cells (or embryonic carcinomacells), early postimplantation embryos and primordial germ cells(Jähaner, D. and Jaenish, R., “DNA Methylation in Early MammalianDevelopment,” In DNA Methylation: Biochemistry and BiologicalSignificance, Razin, A. et al., eds., Springer-Verlag (1984) pp.189-219; Razin, A., and Cedar, H., “DNA Methylation and Embryogenesis,”in DNA Methylation: Molecular Biology and Biological Significance,Jost., J. P. et al, eds. Birkhäuser Verlag, Basel, Switzerland (1993)pp. 343-357; Chaillet, J. R. et al, Cell 66:77-83 (1991); and Li, E.“Role of DNA Methylation in Development,” in Genomic Imprinting:Frontiers in Molecular Biology, Reik, W. and Sorani, A. eds., IRL Press,Oxford (1997) pp. 1-20). The expression of both Dnmt3a and Dnmt3b inmouse embryos, adult tissues and ES cells was examined. The resultsindicate that two Dnmt3a transcripts, 9.5 kb and 4.2 kb, are present inembryonic and adult tissue RNA. The 4.2 kb transcript, corresponding tothe size of the full length cDNA, was expressed at very low levels inmost tissues, except for the E11.5 embryo sample (FIG. 6A). A single 4.4kb Dnmt3b transcript is detected in embryo and adult organ RNAs, withrelatively high levels in testes and E11.5 embryo samples (FIG. 6A).Interestingly, both genes are expressed at much higher levels in EScells than in adult tissues (FIG. 6B), and their expression decreaseddramatically upon differentiation of ES cells in culture (FIG. 6C). Inaddition, Dnmt3a and Dnmt3b expression levels are unaltered inDnmt1-deficient ES cells (FIG. 6C), suggesting that regulation of Dnmt3aand Dnmt3b expression is independent of Dnmt1.

These results suggest that both Dnmt3a and Dnmt3b are expressedspecifically in ES cells and E11.5 embryo and/or testes. The expressionin the E11.5 embryo and testes may correlate with the presence ofdeveloping or mature germ cells in these tissues. Therefore, theexpression pattern of Dnmt3a and Dnmt3b appears to correlate well withde novo methylation activities in development.

For the RNA expression analysis of human DNMT3 genes, polyA+ RNA blotswere hybridized using DNMT3A and DNMT3B cDNA fragments as probes.Results indicate that DNMT3A RNA was expressed ubiquitously and wasreadily detected in most tissues examined at levels slightly lower thanDNMT1 RNA (Fig.X). Three major DNMT3A transcripts, approximately 4.0,4.4, and 9.5 kb, were detected. The relative expression level of thetranscripts appeared to vary from tissue to tissue. Transcripts ofsimilar sizes were also detected in mouse tissues. Results utilizingDNMT3B cDNA probes indicate that transcripts of about 4.2 kb wereexpressed at much lower levels in most tissues, but could be readilydetected in the testis, thyroid and bone marrow (FIG. 9). Sequenceanalyses of different cDNA clones indicate the presence of alternativelyspliced transcripts, although the size differences between thesetranscripts are too small to be detected by Northern analysis.

Hypermethylation of tumor suppressor genes is a common epigenetic lesionfound in tumor cells (Laird, P. W. & Jaenisch, R., Ann. Rev. Genet.30:441-464 (1996); Baylin, S. B., Adv. Cancer Res. 72:141-196 (1998)).To investigate whether DNMT3A and DNMT38 am abnormally activated intumor cells, DNMT3 RNA expression was analyzed in several tumor celllines by Northern blot hybridization. Results demonstrated that DNMT3Awas expressed at higher levels in most tumor cell lines examined. (FIG.10). As in the normal tissues, three different size transcripts werealso detected in tumor cells. The ratio of these transcripts appeared tobe variable in different tumor cell lines. DNMT3B expression wasdramatically elevated in most tumor cell lines examined though it wasexpressed at very low levels in normal adult tissues (FIG. 10). Theexpression levels of both DNMT3A and DNMT3B appear to be comparable andproportional to that of DNMT1.

The murine Dnmt3a and Dnmt3b genes are highly expressed inundifferentiated ES cells, consistent with their potential role in denovo methylation during early embryonic development. Additionally, bothgenes are highly expressed in early embryos. Differences in theirexpression patterns in adult tissues in both human and mice suggest thateach gene may have a distinct function in somatic tissues and maymethylate different genes or genomic sequences. The elevated expressionof DNMT3 genes in human tumor cell lines suggests that the DNMT3 enzymemay be responsible for de novo methylation of CpG islands in tumorsuppressor genes during tumor formation.

Example 4 Methyltransferase Activity Assay

In order to demonstrate DNA cytosine methyltransferase activity, thepolypeptides of the invention were expressed and purified fromrecombinant host cells for use in in vitro assays.

Infected insect Sf21 cells and NIH3T3 cells were homogenized byultrasonication in lysis solution (20 mM Tris-HCl, pH7.4, 10 mM EDTA,500 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1 ug/ml leupeptin, 10ug/ml TPCK, 10 ug/ml TLCK) and cleared by centrifugation at 100,000 gfor 20 min.

The methyltransferase enzyme assay was carried out as describedpreviously (Lei, H. et al., Development 122:3195-3205 (1996)). DNAsubstrates used in the assays include: poly (dI-dC), poly (dG-dC)(Pharmacia Biotech), lambda phage DNA (Sigma), pBluescriptIISK(Stratagene, Calif.), pMu3 plasmid, which contains tandem repeats of 535bp RsaI-RsaI fragment of MMLV LTR region in pUC9, and oligonucleotides.The oligonucleotide sequences utilized include: #1,5′-AGACMGGTGCCAGMGCAGCTGAGCMGGATC-3′, #2,5′-GATCMGGCTCAGCTGMGCTGGCACMGGTCT-3′, #3,5′-AGACCGGTGCCAGCGCAGCTGAGCCGGATC-3′, and #4,5′-GATCCGGCTCAGCTGCGCTGGCACCGGTCT-3′. (M represents 5-methylcytosine)

These sequences are the same as described in a previous study (Pradhan,S. et al., Nucleic Acids Res. 25:4666-4673 (1997)). Oligonucleotideswere synthesized and purified by polyacrylamide gel electrophoresis(PAGE). To make double strand oligonucleotides, equimolar amounts of thetwo complimentary oligonucleotides were heated at 94° C. for 10 min.,mixed, incubated at 78° C. for 1 hr and cooled down slowly at roomtemperature. The annealing products were quantified for the yield ofdouble-stranded oligonucleotides (dsDNA) by PAGE and methylene bluestaining. In all cases, the yield of dsDNA was higher than 95%. ThedsDNA of #1 and #2 were used as ‘fully’ methylated substrates, dsDNA of#1 and #4 as the hemi-methylated substrates, and dsDNA of #3 and #4 asunmethylated substrates.

For Southern analysis of the methylation of retrovirus DNA, 2 ug ofpMMLV8.3, an 8.3 kb Hind III fragment of Moloney murine leukemia viruscDNA in pBluescriptIISK, was methylated in vitro for 15 hrs under thesame reaction conditions described above except that 160 uM of cold SAMwas used instead of ³H-methyl SAM. Then, an equal volume of the solutioncontaining 1% SDS, 400 mM NaCl, and 0.2 mg/ml Proteinase K was added,and the sample was incubated at 37° C. for 1 hr. After phenol/chloroformextraction, DNA was precipitated with ethanol, dried and dissolved in TEbuffer. This procedure was repeated 5 times. An aliquot of DNA waspurified after the first, third and fifth reaction, digested with Hpa IIor Msp I in combination with Kpn I for 16 hrs, separated on 1% agarosegels, blotted and hybridized to the pMu3 probe.

In a standard methyltransferase assay, enzyme activity was detected withprotein extracts from Sf21 cells overexpressing Dnmt3a and Dnmt3bpolypeptides. Similar to the results obtained with the Dnmt1polypeptide, the overexpressed Dnmt3 proteins were able to methylatevarious native and synthetic DNA substrates, among which poly(dI-dC)consistently gave rise to the highest initial velocity (FIG. 7 a). Ananalysis of the methylation of Hpa II sites in retroviral DNA by theseenzymes was also performed. An MMLV full length cDNA was methylated for1-5 times by incubation with protein extract from control Sf21 cells orSf21 cells infected with baculoviruses expressing Dnmt1, Dnmt3a orDnmt3b polypeptides. The Hpa II/Msp I target sequence, CCGG, isresistant to the Hpa 11 restriction enzyme, but sensitive to Msp Idigestion when the internal C is methylated, and the restriction sitebecomes resistant to Msp I digestion when the external C is methylated(Jentsch, S. et al. Nucleic Acids Res. 9:2753-2759 (1981)). Both Dnmt3aand Dnmt3b polypeptides could methylate multiple Hpa 11 sites in the 3′LTR regions of the MMLV DNA, as indicated by the presence of HpaII-resistant fragments, though less efficiently than Dnmt1 polypeptide(FIG. 7 b). Significantly, even after five consecutive rounds of invitro methylation, the viral DNA was completely digested by Msp 1. Thisresult indicates that both Dnmt3a and Dnmt3b polypeptides methylatepredominantly the internal cytosine residues, therefore, CpGs.Previously it was shown that the same region of the proviral DNA wasefficiently methylated in Dnmt1 null ES cells infected by the MMLV virus(Lei. H. et al., Development 122:3195-3205 (1996)).

FIG. 7A shows ³H-methyl incorporation into different DNA substrates(poly (dI-dC), poly (dG-dC) (squares), lambda phage DNA (circles),pBlucscriptIISK (triangles), and pMu3 (diamonds)) when incubated withprotein extracts of Sf21 cells expressing Dnmt1, Dnmt3a, or Dnmt3b1.FIG. 7B shows Southern blot analysis of the in vitro methylation ofuntreated pMMLV DNA (lanes 1-3) and pMMLV DNA incubated with MT1 (lane4-10), MT3a (lanes 11-15), MT3β (lanes 16-20) or control Sf21 (lanes21-25) extracts that were digested with Kpn I(K), Kpn I and Msp I (K/M)or Kpn I and Hpa II (K/H). Restriction enzyme digested samples were thensubjected to Southern blot analysis using the pMu3 probe.

Dnmt1 protein appears to function primarily as a maintenancemethyltransferase because of its strong preference for hemimethylatedDNA and direct association with newly replicated DNA (Leonhardt, H. etal., Cell 71:865-873 (1992)). To determine whether Dnmt3a and Dnmt3bpolypeptides show any preference for hemimethylated DNA overunmethylated DNA, a comparison was done to examine the methylation rateof unmethylated versus hemimethylated oligonucleotides. Gel-purifieddouble stranded oligonucleotides were incubated with protein extracts ofSf21 cells expressing Dnmt1, Dnmt3a, Dnmt3b1, Dnmt3b2 or NIH3T3 cellextract (unmethylated substrates (open circles), hemi-methylatedsubstrates (half black diamonds) or completely methylated substrates(closed squares)). While baculovirus-expressed Dnmt1 polypeptide or 3T3cell extract showed much higher activities when hemimethylated DNA wasused as a substrate, Dnmt3a, Dnmt3b 1 and Dnmt3b2 polypeptides showed nodetectable preference for hemimethylated DNA (FIG. 8).

1. An isolated nucleic acid molecule comprising a polynucleotideselected from the group consisting of: a. a polynucleotide sequenceencoding a polypeptide comprising amino acids from about 1 to about 908in SEQ ID NO: 5; b. a polynucleotide sequence encoding a polypeptidecomprising amino acids from about 1 to about 859 in SEQ ID NO:6; c. apolynucleotide sequence encoding a polypeptide comprising amino acidsfrom about 1 to about 912 in SEQ ID NO:7; d. a polynucleotide sequenceencoding a polypeptide comprising amino acids from about 1 to about 853in SEQ ID NO:8; e. a polynucleotide sequence that is at least 90%identical to the polynucleotide sequence of (a), (b), (c) or (d); and f.a polynucleotide sequence complementary to the polynucleotide sequenceof (a), (b), (c), (d) or (e), wherein said polypeptide is capable ofmethylating DNA in an in vitro assay.
 2. (canceled)
 3. A method ofmaking a recombinant vector comprising inserting an isolated nucleicacid molecule of claim 1 into a vector selected from a group consistingof: a. a DNA vector; and b. an RNA vector.
 4. A recombinant vectorcomprising the isolated nucleic acid molecule of claim
 1. 5. A method ofmaking a recombinant host cell comprising introducing the recombinantvector of claim 4 into a host cell.
 6. A recombinant host cellcomprising the vector of claim
 4. 7. A method for producing a de novoDNA cytosine methyltransferase polypeptide, comprising culturing therecombinant host cell of claim 6 under conditions such that saidpolypeptide is expressed and recovering said polypeptide.
 8. An isolatedoligonucleotide probe or primer comprising polynucleotides selected fromthe group consisting of: a. at least 50 contiguous nucleotides of SEQ IDNO:1, provided that said nucleotides are not AA052791(SEQ ID NO: 9);AA111043(SEQ ID NO:10); AA154890(SEQ ID NO:11); AA240794(SEQ ID NO:12);AA756653(SEQ ID NO:13); W58898(SEQ ID NO:14); W59299(SEQ ID NO:15);W91664(SEQ ID NO:16); W91665(SEQ ID NO: 17); and b. a nucleotidesequence complementary to a nucleotide sequence in (a).
 9. An isolatedoligonucleotide probe or primer comprising polynucleotides selected fromthe group consisting of: a. at least 30 contiguous nucleotides of SEQ IDNO:2, provided that said nucleotides are not AA116694 (SEQ ID NO:18);AA119979 (SEQ ID NO:19); AA177277 (SEQ ID NO:20); AA210568 (SEQ IDNO:21); AA399749 (SEQ ID NO:22); AA407106 (SEQ ID NO:23); AA575617 (SEQID NO:24); and b. a nucleotide sequence complementary to a nucleotidesequence in (a).
 10. An isolated oligonucleotide probe or primercomprising polynucleotides selected from the group consisting of: a. atleast 100 contiguous nucleotides of SEQ ID NO:3, provided that saidnucleotides are not AA004310 (SEQ ID NO:25); AA004399 (SEQ ID NO:26);AA312013 (SEQ ID NO:27); AA355824 (SEQ ID NO:28); AA533619 (SEQ IDNO:29); AA361360 (SEQ ID NO:30); AA364876 (SEQ ID NO:31); AA503090 (SEQID NO:32); AA533619 (SEQ ID NO:33); AA706672 (SEQ ID NO:34); AA774277(SEQ ID NO:35); AA780277 (SEQ ID NO:36); H03349 (SEQ ID NO:37); H04031(SEQ ID NO:38); H53133 (SEQ ID NO:39); H53239 (SEQ ID NO:40); H64669(SEQ ID NO:41); N26002 (SEQ ID NO:42); N52936 (SEQ ID NO:43); N88352(SEQ ID NO:44); N89594 (SEQ ID NO:45); R19795 (SEQ ID NO:46); R47511(SEQ ID NO:47); T50235 (SEQ ID NO:48); T78023 (SEQ ID NO:49); T78186(SEQ ID NO:50); W22886 (SEQ ID NO:51); W67657 (SEQ ID NO:52); W68094(SEQ ID NO:53); W76111 (SEQ ID NO:54); Z38299 (SEQ ID NO:55); Z42012(SEQ ID NO:56); G06200(SEQ ID NO:74); and b. a nucleotide sequencecomplementary to a nucleotide sequence in (a). 11-12. (canceled)
 13. Amethod for in vitro de novo methylation of DNA; comprising: a.contacting said DNA with a de novo DNA cytosine methyltransferasepolypeptide encoded by the nucleic acid molecule of claim 1; b.providing an appropriately buffered solution with substrate andcofactor; and c. purifying said DNA. 14-24. (canceled)
 25. The nucleicacid molecule of claim 1, wherein said polynucleotide is that of part(a).
 26. The nucleic acid molecule of claim 1, wherein saidpolynucleotide is that of part (b).
 27. The nucleic acid molecule ofclaim 1, wherein said polynucleotide is that of part (c).
 28. Thenucleic acid molecule of claim 1, wherein said polynucleotide is that ofpart (d).
 29. The nucleic acid molecule of claim 1, wherein saidpolynucleotide is that of part (e).
 30. The nucleic acid molecule ofclaim 1, wherein said polynucleotide is that of part (f).
 31. Anisolated nucleic acid molecule comprising a polynucleotide selected fromthe group consisting of: a. a polynucleotide sequence encoding mouseDnmt3a polypeptide contained in ATCC Deposit No. 209933; b. apolynucleotide sequence encoding mouse Dnmt3b polypeptide contained inATCC Deposit No. 209934; c. a polynucleotide sequence encoding humanDNMT3A polypeptide contained in ATCC Deposit No. 98809; d. apolynucleotide sequence encoding human DNMT3B polypeptide contained inATCC Deposit No. 326637; e. a polynucleotide sequence at least 90%identical to the polynucleotide sequence of (a), (b), (c) or (d); and f.a polynucleotide sequence complementary to the polynucleotide sequenceof (a), (b), (c), (d) or (e), wherein said polypeptide is capable ofmethylating DNA in an in vitro assay.
 32. The nucleic acid molecule ofclaim 31, wherein said polynucleotide is that of part (a).
 33. Thenucleic acid molecule of claim 31, wherein said polynucleotide is thatof part (b).
 34. The nucleic acid molecule of claim 31, wherein saidpolynucleotide is that of part (c).
 35. The nucleic acid molecule ofclaim 31, wherein said polynucleotide is that of part (d).
 36. Thenucleic acid molecule of claim 31, wherein said polynucleotide is thatof part (e).
 37. The nucleic acid molecule of claim 31, wherein saidpolynucleotide is that of part (f).
 38. An isolated nucleic acidmolecule comprising a polynucleotide at least 95% identical to apolynucleotide selected from the group consisting of: a. apolynucleotide sequence encoding a polypeptide comprising amino acidsfrom about 1 to about 908 in SEQ ID NO:5; b. a polynucleotide sequenceencoding a polypeptide comprising amino acids from about 1 to about 859in SEQ ID NO:6; c. a polynucleotide sequence encoding a polypeptidecomprising amino acids from about 1 to about 912 in SEQ ID NO:7; d. apolynucleotide sequence encoding a polypeptide comprising amino acidsfrom about 1 to about 853 in SEQ ID NO:8; and e. a polynucleotidesequence complementary to the polynucleotide sequence of (a), (b), (c)or (d), wherein said polypeptide is capable of methylating DNA in an invitro assay.
 39. The nucleic acid molecule of claim 38, wherein saidpolynucleotide is that of part (a).
 40. The nucleic acid molecule ofclaim 38, wherein said polynucleotide is that of part (b).
 41. Thenucleic acid molecule of claim 38, wherein said polynucleotide is thatof part (c).
 42. The nucleic acid molecule of claim 38, wherein saidpolynucleotide is that of part (d).
 43. The nucleic acid molecule ofclaim 38, wherein said polynucleotide is that of part (e).
 44. Anisolated nucleic acid molecule comprising a polynucleotide selected fromthe group consisting of a. SEQ ID NO:1; b. SEQ ID NO:2; c. SEQ ID NO:3;d. SEQ ID NO:4; e. a polynucleotide sequence that is at least 90%identical to the polynucleotide sequence of (a), (b), (c) or (d); and f.a polynucleotide sequence complementary to the polynucleotide sequenceof (a), (b), (c), (d) or (e), wherein said polynucleotide encodes apolypeptide capable of methylating DNA in an in vitro assay.
 45. Thenucleic acid molecule of claim 44, wherein said polynucleotide is thatof part (a).
 46. The nucleic acid molecule of claim 44, wherein saidpolynucleotide is that of part (b).
 47. The nucleic acid molecule ofclaim 44, wherein said polynucleotide is that of part (c).
 48. Thenucleic acid molecule of claim 44, wherein said polynucleotide is thatof part (d).
 49. The nucleic acid molecule of claim 44, wherein saidpolynucleotide is that of part (e).
 50. The nucleic acid molecule ofclaim 44, wherein said polynucleotide is that of part (f).